Novel method for cloning variable domain sequences of immunological gene repertoire

ABSTRACT

The present invention relates to a non-PCR (polymerase chain reaction) process, particularly a transcription-based amplification method, for amplifying and cloning sequences containing a variable domain sequence such as an immunoglobulin variable domain sequence from the immunological gene repertoire. The present invention comtemplates the expression of antibody library in either in an in vivo expression vector or in an in vitro transcription/translation system. Isolation of a gene coding for a receptor having the ability to bind a preselected ligand and receptors produced by the gene isolated by the method is also contemplated.

TECHNICAL FIELD

[0001] The present invention relates to methods for amplifying andcloning variable region or domain sequences of the immunological genes,generating libraries of immunological gene repertoire and isolating agene coding for an antigen-combining molecule such as antibody orimmunoglobulin.

BACKGROUND

[0002] A dozen or so monoclonal antibodies have been approved by theFood and Drug Administration (FDA) as human therapeutics includingOrthoclone OKT3 for allograft rejection, ReoPro (abciximab) for adjuncttreatment of percutaneous coronary intervention (PCI) including balloonangioplasty, atherectomy and stent placement, Rituxan for Non-Hodgkin'slymphoma, Simulet and Zenapax for organ rejection prophylaxis, Remicadefor Rheumatoid arthritis and Crohn's disease, Synagis for respiratorysyncytial virus (RSV), Herceptin for metastatic breast cancer, Mylotargfor acute myeloid leukemia and Campath for chronic lymphocytic leukemia,etc. These therapeutic antibodies can be divided into three majorcategories: murine monoclonal antibodies (Orthoclone OKT3); chimericmonoclonal antibodies (ReoPro, Rituxan, Simulet, and Remicade); andCDR-grafted monoclonal antibodies (Zenapax, Synagis, Herceptin,Mylotarg, and Campath). A murine monoclonal antibody is a mouseantibody; a chimeric antibody contains antibody of two or more speciesof animal, such as human and mouse; while CDR-grafted antibodies havelower amounts of foreign protein, generally in the complementaritydetermining region (CDR), thus the framework is human and the CDR are ofmouse origin. In the above clinically approved antibodies, the non-humanportion of the antibody derived is from a mouse antibody.

[0003] The mouse portion of the murine, chimeric or even CDR-graftedantibodies would elicit an immune response and associated side effectswhen administrated to a human, such as HAMA (human anti-mouse antibody)or HACA (human anti-chimeric antibody) responses. Thus, therapeuticantibody development is best suited with totally or 100% humanantibodies.

[0004] There are two approaches in making human antibodies. One approachuses a human-mouse system such as the XenoMouse technology of Abgenix(Fremont, Calif.) or the HuAbMouse technology of Medarex, Inc.(Princeton, N.J.), wherein the host mouse immunoglobulin genes areinactivated and most of the human immunoglobulin genes are incorporatedinto the mouse to produce totally human antibodies in response toantigen stimuli in the mouse. Some of the difficulties in producingmonoclonal antibodies with the human-mouse methodology include geneticinstability, smaller antigenic specificities due to tolerancerestriction of certain antibodies in a live animal, low throughput inaccess and screening of the in vivo antibody repertoire which can onlybe accessed via immunization with a selection on the basis of bindingaffinity and low production capacity.

[0005] The other approach is to generate libraries of antibody genes bycloning. Often the target genes are amplified prior to cloning.

[0006] There are a number of methods in the field of amplifying specifictarget nucleic acid sequences of interest. The polymerase chain reactionmethod (PCR), as described by Mullis et al., (see U.S. Pat. Nos.4,683,195, 4,683,202, and 4,800,159; and European Patent ApplicationNos. 86302298.4, 86302299.2, and 87300203.4, and Methods in Enzymology,Volume 155, 1987, pp. 335-350), is one of the most prominent methods.PCR involves the use of a pair of specific oligonucleotides as primersfor the two complementary strands of the double-stranded DNA containingthe target sequence. The primers are chosen to hybridize at the ends ofeach of the complementary target strands, 3′ of the target sequence.Template-dependent DNA synthesis, on each strand, can then be catalyzedusing a thermostable DNA polymerase in the presence of the appropriatereagents. A thermal cycling process is required to form specific hybridsprior to synthesis and then to denature the double stranded nucleic acidformed by synthesis. Repeating the cycling process geometricallyamplifies the target sequence.

[0007] A PCR method employing a reverse transcription step is also usedwith an RNA target using RNA-dependent DNA polymerase to create a DNAtemplate. The PCR method has been coupled to RNA transcription byincorporating a promoter sequence into one of the primers used in thePCR reaction and then, after amplification by the PCR method, using thedouble-stranded DNA as a template for the transcription ofsingle-stranded RNA. (see, e.g., Murakawa et al., DNA 7:287-295 (1988)).The PCR method has been applied to the amplification and cloning of thevariable domain sequences of immunoglobulin or antibody genes (U.S. Pat.No. 6,291,158 to Winter et al. and U.S. Pat. No. 6,291,161 to Lerner etal.).

[0008] There are, however, several non-PCR-based amplification methodsthat can be used for amplifying specific target genes. One types of thenon-PCR-based amplification methods include multiple cycles ofDNA-dependent RNA polymerase-driven RNA transcription amplification orRNA-directed DNA synthesis and transcription to amplify DNA or RNAtargets (see, e.g., Burg et al., WO 89/01050; Gingeras et al., WO88/10315; Kacian and Fultz, EPO Application No. 89313154; Davey andMalek, EPO Application No. 88113948.9; Malek et al., WO91/02818 and U.S.Pat. No. 5,130,238; Davey et al., U.S. Pat. Nos. 5,409,818; 5,466,586;5,554,517 and 6,063,603; Eberwine et al., U.S. Pat. No. 5,514,545; Linet al., U.S. Pat. No. 6,197,554; and Kacian et al., U.S. Pat. No.5,888,779).

[0009] Another type of amplification method uses a ligase chain reaction(LCR) as described in European Patent Publication No. 320,308. Thismethod requires at least four separate oligonucleotides, two of whichhybridize to the same nucleic acid template so their respective 3′ and5′ ends are juxtaposed for ligation. The hybridized oligonucleotides arethen ligated forming a complementary strand on the nucleic acidtemplate. The double-stranded nucleic acid is then denatured, and thethird and fourth oligonucleotides are hybridized with the first andsecond oligonucleotides that were joined together. The third and fourtholigonucleotides are then ligated together. Amplification is achieved byfurther cycles of hybridization, ligation, and denaturation.

[0010] Another amplification method uses Qβ replicase (Qβ) method asdescribed in PCT Publication Ser. No. 87/06270 and U.S. Pat. No.4,786,600 that uses a specific RNA probe which is capable of specifictranscription by a replicase enzyme. The method requires the design andsynthesis of RNA probes with replicase initiation sites.

[0011] Another type of amplification uses palindromic probes asdescribed in EPO Publication Nos. 0427073A and 0427074A. The palindromicprobe forms a hairpin with a nucleic acid target sequence. The probecontains a functional promoter located in the hairpin region from whichRNA transcripts are produced.

[0012] There are also several versions of a strand displacementamplification method that uses one strand of DNA to displace same strandDNA sequences hybridized to their complementary DNA sequences togenerate many copies of the target DNA sequences under isothermalconditions.

[0013] Walker et al., Proc. Nati. Acad. Sci. U.S.A., 89:392-396 (January1992), Walker et al., Nucl. Acids Res. 20:1691-1696 (1992), EuropeanPatent Application No. EP 0 497272, and European Patent Application No.EP 0 500 224, describe an oligonucleotide-driven amplification methodusing a restriction endonuclease. The restriction endonuclease nicks theDNA/DNA complex to enable an extension reaction and, therefore,amplification.

[0014] Becker et al., EPO Application No. 88306717.5, describe anamplification method in which a primer is hybridized to a nucleic acidsequence and the resulting duplex cleaved prior to the extensionreaction and amplification.

[0015] Dattagupta et al. described another version of the stranddisplacement amplification method by using a nucleic acid polymeraselacking 5′ exonuclease activity and a set of oligonucleotide primers tocarry out isothermal amplification without requiring exonucleaseactivity or restriction endonuclease activity (U.S. Pat. No. 6,214,587).

[0016] Another amplification method is rolling circle amplification. Themethod involves insertion of a nucleic acid molecule of interest in alinear vector to form a circular vector where one strand is continuousand the other strand is discontinuous. The continuous strand of thecircular vector is then amplified by rolling circle replication,amplifying the inserted nucleic acid molecule in the process. Theamplification is rapid and efficient since it involves a single,isothermal reaction that replicates the vector sequences exponentially(U.S. Pat. No. 6,287,824 to Lizardi).

[0017] A related amplification method using a similar approach is termedramification extension amplification (RAM), U.S. Pat. No. 5,942,391 toZhang et al. The RAM method involves hybridizing a target nucleic acidto several non-overlapping oligonucleotide probes that hybridize toadjacent regions in the target nucleic acid, the probes being referredto as capture/amplification probes and amplification probes,respectively, in the presence of paramagnetic beads coated with a ligandbinding moiety. Through the binding of a ligand attached to one end ofthe capture/amplification probe and the specific hybridization ofportions of the probes to adjacent sequences in the target nucleic acid,a complex comprising the target nucleic acid, the probes and theparamagnetic beads is formed. The probes may then ligate together toform a contiguous ligated amplification sequence bound to the beads,which complex may be denatured to remove the target nucleic acid andunligated probes.

[0018] Attempts to clone variable domain sequences of the immunologicalgenes into an antibody framework vector and expressing the antibodies ina host cell such as in a phage using PCR have been described (U.S. Pat.No. 6,291,158 to Winter, et al.; and U.S. Pat. No. 6,291,161 to Lerner,et al.). Some of the difficulties in employing that PCR amplificationscheme are that PCR amplification efficiency is dependent on both theprimer and the template sequences. Certain sequences are preferentiallyamplified with other sequences being under-amplified or not amplifiedleading to under representation of the diversity of the resultingantibody libraries. An example of the limitations encountered when usingPCR to clone a library is provided in Gao et al., Proc. Natl. Acad. Sci.(1999) 96:6025-6030.

[0019] The size of the human antibody repertoire is estimated to be onthe order of 10⁶ to 10⁸ different antigen specificities. Exceptionallarger numbers of specificities of the human antibodies can be generatedby in vitro construction of V_(H) and/or V_(L) libraries by randomrecombination and shuffling, and saturation mutagenesis of the V_(H) andV_(L) DNA homologs.

[0020] One of the potential benefits of constructing human antibodylibraries is to obviate the need for immunization by the generation ofhighly diverse “generic” human antibody libraries. In certain cases,very specialized human antibody libraries such as human antibodylibraries made by using blood cells of cancer patients or blood cells ofpatients with autoimmune diseases such as rheumatoid arthritis,psoriasis, etc. may contain human antibodies with very high avidity andspecificity for that particular diseases. Another benefit of havinghuman antibody libraries is that they permit iterative cycles ofmutagenesis or random recombination of the V_(H) and V_(L) generepertoire to further optimize the specificity, affinity or catalyticproperties of the immunoglobulins or their derivative antibodies such asF_(ab) and scF_(v) fragments.

BRIEF SUMMARY OF THE INVENTION

[0021] The present invention relates to methods for amplifying andcloning variable region or domain sequences of immunological genes,generating libraries of immunological gene repertoire and isolating agene coding for an antigen-combining molecule such as antibody orimmunoglobulin. The present invention employs a non-PCR amplificationprocess such as DNA-dependent RNA polymerase driven RNAtranscription-based amplification methods, strand displacementamplification methods, ligase chain reaction, rolling circleamplification, ramification amplification methods, and replicase drivenamplification methods.

[0022] The present invention specifically contemplates and details atranscription-based amplification scheme for amplifying and cloningsequences containing a sequence encoding a variable domain sequence suchas an immunoglobulin variable domain sequence. The transcription-basedamplification (TCA) process is based on transcriptional amplification byDNA-dependent RNA polymerase from DNA sequences containing a promotersequence for the binding of RNA polymerase and the initiation of RNAtranscription activity as shown in FIG. 1. Unlike the two-fold per cycleamplification rate of a PCR-based reaction, the RNA polymerase drivenRNA transcription based amplification process provides up to, per oneround of RNA transcription, two thousand-fold amplification of thedesired starting materials, especially to the immunoglobulin variabledomain sequences. Following TCA, the resulting double stranded DNA iscloned in a suitable vector. That process non-selectively, outside ofthe specificity imposed by the primers, which are of design choice andbased on the molecules to be cloned, amplifies all suitable targetsequences thereby enriching rare targets or targets generally unsuitablefor amplification and cloning using other amplification methods andensuring suitable levels of target for cloning.

[0023] The advantages of using RNA transcriptional amplification ratherthan PCR amplification are: firstly, single copy messenger RNA (mRNA)can be increased up to 2000-fold in one round of amplification withproofreading activity. Secondly, the RNA transcriptional amplificationis linear and does not result in preferential amplification, which is amajor problem associated with PCR-based amplification reactions.Thirdly, the RNA transcriptional amplification process can be applied toamplify multiple sequences simultaneously with a capacity of amplifyingat least more than ten to twenty sequences at a time in a singlereaction, while the PCR-based amplification has limited ability formultiplexing with a typical limit of amplifying less than ten, mostoften less than five sequences in a single group reaction (Gao et al.,supra). The RNA transcriptional amplification method has often been usedto amplify or to reproduce the entire mRNA transcriptome from a singleneuron cell or 20 to 50 cancer cells. (Lin et al., Nucl. Acids Res.27:4585-4598, 1999). Also, the resulting amplified mRNA products, insome cases full-length sequences, can be used for furtherpeptide/protein synthesis directly in an in vitrotranscription/translation system such as the PROfusion and the ribosomedisplay technologies as further discussed below in the presentinvention.

[0024] One can use the RNA transcriptional amplification process totranscriptionally amplify the mRNA transcripts of a gene family by morethan one billion-fold and subject the resultant amplified mRNAtranscripts of the gene family directly to translation intopeptide/protein products in vitro or in vivo for antigen-antibodyrelated applications. Alternatively, one can convert the amplified mRNAsinto double-stranded cDNAs with appropriate restriction enzyme sites forfurther cloning into an expression-competent vector in a host cell, suchas in a phage display system.

[0025] The present invention provides a novel non-PCR-basedamplification method for amplifying and cloning a larger population ofthe immunological repertoire for immunoglobulin genes and receptorshaving a preselected activity against immunogens, ligands, smallmolecules or macromolecules, thereby overcoming the before-mentionedinadequacies of preferential amplification of the PCR-basedamplification technique.

[0026] In one embodiment, a gene library is generated or synthesized forthe immunoglobulin light kappa chain variable region (V_(K)) generepertoire of a vertebrate such as a human being. In preferredembodiments, the V_(K) gene library contains at least about 10³,preferably at least about 10⁴, more preferably at least about 10⁵, andmost preferably at least about 10⁶ different V_(K) DNA homologs,although higher diversity amounts of 10⁷-10¹¹ clones are possiblebecause of the library properties inherent when using filamentous phage.

[0027] Methods for evaluating the diversity of a gene repertoire arewell known to one skilled in the art.

[0028] The V_(K) gene library can be synthesized by either of twomethods, depending on the starting material, mRNAs or genomic DNAs.

[0029] In one embodiment, the mRNAs of a tissue or cell of immune systemorigin, such as blood cells, are used as the source of immunologicalgenes. The mRNAs of the immunoglobulin genes are reversed transcribed byspecific V_(K) antisense primers operatively linked with a promotersequence for a DNA-dependent RNA polymerase such as T7 promoter (the RNApromoter-linked primer). The RNA promoter-linked primer for the V_(K)genes is situated in the conserved region adjacent to the variabledomain of the V_(K) genes, such as the J region in the constant regionof the kappa light chain. The resulting cDNAs are made intodouble-stranded (ds) cDNAs by double stranding reactions with senseV_(K) primers situated in the relatively conserved region in thevariable region of the V_(K) genes. The ds cDNAs are then transcribed bythe DNA-dependent RNA polymerase such as T7 polymerase into hundreds tothousands copies of antisense RNA transcripts. The amplified RNAtranscripts can be amplified again. The antisense RNA transcripts arereverse transcribed into sense DNAs (sDNAs) by the sense V_(K) primers,the single stranded sDNAs are made into ds DNAs with the RNApromoter-linked primers. The resulting ds DNAs can be transcribed intohundreds to thousands of copies of RNA transcripts again by theDNA-dependent RNA polymerase. With two rounds of transcriptionamplification, the original mRNAs of immunoglobulin genes are copiedinto hundreds of thousands to millions of copies of antisense RNAtranscripts complementary to the original sequences. The above processcan be repeated or cycled a few more times, if needed. The resulting RNAtranscripts can be easily converted into ds DNAs by techniques known inthe art and the resulting ds DNAs are ready for further cloning and/orexpression of the antigen-combining molecules on in vitro transcriptionand translation or in an expression vector in a host cell such as alambda phage.

[0030] In another embodiment, genomic DNAs from an immunological tissueor cells such as blood cells or other stimulated immunological cellswith rearranged immunoglobulin genes, are used as the cloning source forthe V_(K) genes. The variable region of the immunoglobulin genes arecopied only once into ds DNAs by the two primers as discussed in theprevious embodiments, the ds DNAs are amplified by a similar RNAtranscription based amplification process discussed in the previousembodiments and so on.

[0031] In another embodiment, a similar approach and method to the V_(K)gene library cloning processes as discussed in the previous embodimentsare employed to generate or to synthesize the immunoglobulin light chainlambda variable region (V_(λ)) gene repertoire of a vertebrate such as ahuman being. In preferred embodiments, the V_(λ) gene library containsat least about 10³, preferably at least about 10⁴, more preferably atleast about 10⁵, and most preferably at least 10⁶ different V_(λ) DNAhomologs, although higher diversity amounts of 10⁷ to about 10¹¹ clonesare possible because of the library properties inherent when usingfilamentous phage.

[0032] In another embodiment, a similar approach and method to the V_(K)gene library cloning processes as discussed in the previous embodimentsare employed to generate or synthesize the immunoglobulin heavy chainvariable region (V_(H)) gene repertoire of a vertebrate such as a humanbeing. In preferred embodiments, the V_(H) gene library contains atleast about 10³, preferably at least about 10⁴, more preferably at leastabout 10⁵, and most preferably at least 10⁶ different V_(H) DNAhomologs, although higher diversity amounts of 10⁷ to more than 10¹¹clones are possible because of the library properties inherent whenusing filamentous phage.

[0033] Additionally, the size and diversity of the library of proteinscan be increased by introducing a modifying step wherein the nucleicacids are changed to yield new proteins. Those new proteins can yieldproteins that have the same antigen specificity with the same ordiffering properties. For example, effector functions, such as bindingand activating complement, can vary between the parent protein and themodified derivative protein. Alternatively, the modified proteins canhave a different antigen specificity. Such modification can be obtainedby mutagenesis, such as a generalized mutagenesis procedure, for exampleusing terminal transferase, or a specific procedure such assite-directed mutagenesis.

[0034] For cloning and expression purposes, the antisense and senseprimers described in the above embodiments are designed to haveappropriate restriction enzyme digestion sites and are fused intoappropriate expression vectors or in vitro transcription and translationframework sequences. The expression of the antigen-combining moleculescan be achieved either via in vitro transcription and translation or byexpression in a host cell through cloning in an expression vector asknown in the art. For in vitro transcription and translation expressionof the immunological genes, the primers are designed to fuseappropriately into a framework sequence, which contains sequencesnecessary for adequate coupling of in vitro transcription andtranslation utilizing methods known in the art.

[0035] The amplified sequences of the variable domains of theimmunological genes such as immunoglobulin genes can be cloned intoantibody framework vectors for expression in host cells such as lambdabacterial phage or mammalian cells. The sequences can also be insertedinto antibody framework sequences useful for in vitro translationcoupling with in vitro transcription. The framework sequences can be ofantibody origin or other framework scaffold molecules useful inpresenting antigen-combining activities of the antibody variableregions.

[0036] The variable domains of the immunoglobulin heavy chain V_(H) andlight chains V_(L) (V_(K) and V_(λ)) can be separately amplified andcloned, expressed and combined. Alternatively, the variable domains ofthe heavy and light chains of the immunoglobulins can be linked with apeptide linker to form single chain F_(v) (scF_(v)) or a single chainantibody.

[0037] An immunological receptor having a preselected activity,preferably catalytic activity, produced by a method of the presentinvention, preferably a V_(H), V_(K), V_(L), F_(ab) and/or scF_(v) asdescribed herein, is also contemplated.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038]FIG. 1 illustrates a schematic representation of a general schemeof transcription based RNA amplification. Briefly, an antisense primeris used to reverse transcribe mRNA into cDNA using a reversetranscriptase. The ds cDNA contains the RNA promoter sequence and istranscribed into RNA transcripts by the RNA polymerase. The amplifiedRNA transcripts can be amplified again by repeating the process. The dscDNAs with appropriate restriction enzyme sites can be cloned intoappropriate expression vector or in vitro transcription/translation unitsequence for further expression in vivo or in vitro. The RNA promotersequence can be linked either with the antisense primer or the senseprimer. The RNA transcripts can be in sense orientation or antisenseorientation dependent on whether the RNA promoter sequence is linked tothe sense or antisense primer. An amplification scheme for amplifyingthe immunoglobulin gene variable regions using mRNAs as the source,employing antisense promoter-linked primers for reverse transcriptionand RNA transcription, and a strand separation to enable making thedouble stranded cDNA.

[0039]FIG. 2 illustrates a schematic diagram of another RNAtranscription based amplification scheme for amplifying theimmunoglobulin gene variable regions using mRNAs as a source, employingantisense promoter-linked primers for reverse transcription and RNAtranscription, and a sense gene-specific primer extension based doublestranding scheme for making double stranded cDNA.

[0040]FIG. 3 illustrates a schematic diagram of another RNAtranscription based amplification scheme for amplifying theimmunoglobulin gene variable regions using mRNAs as the source,employing sense promoter-linked primers for RNA transcription and doublestranding, and antisense primers for reverse transcription. A stopprimer is used in the scheme to prevent over extension of the firststrand cDNA beyond the sense primer site so that the ds RNA promotertemplate can be formed in the primer extension double stranding process.

[0041]FIG. 4 illustrates a schematic diagram of another RNAtranscription based amplification scheme for amplifying theimmunoglobulin gene variable regions using mRNAs as the source,employing terminal transferase for oligonucleotide tailing,promoter-linked primers for double stranding and RNA transcription, andantisense primers for reverse transcription.

[0042]FIG. 5 illustrates a schematic diagram of another RNAtranscription based amplification scheme for amplifying theimmunoglobulin gene variable regions using genomic DNA as the source andemploying antisense promoter-linked primers for RNA transcription.

[0043]FIG. 6 illustrates a schematic diagram of another RNAtranscription based amplification scheme for amplifying theimmunoglobulin gene variable regions using genomic DNA as the source andemploying sense promoter-linked primers for RNA transcription. A stopprimer is used in the scheme to prevent over extension of the antisenseDNA beyond the sense primer site so that the ds RNA promoter templatecan be formed in the primer extension double stranding process.

[0044]FIG. 7 illustrates a schematic diagram for making a V_(H) phagelibrary.

[0045]FIG. 8 illustrates a schematic diagram for making a V_(L) (V_(K)and V_(λ)) phage library.

[0046]FIG. 9 illustrates a schematic diagram for constructing aV_(H)+V_(L) phage library.

[0047]FIG. 10 illustrates a schematic diagram for generating an scF_(v)DNA homologs.

DETAILED DESCRIPTION OF THE INVENTION

[0048] Referring to FIGS. 1 to 10, a novel method for cloning variabledomain sequences of immunological gene repertorie of the presentinvention is illustrated, wherein open bars represent RNA and solid barsrepresent DNA.

[0049] A. Definitions

[0050] To facilitate understanding of the invention, a number of termsare defined below:

[0051] Nucleotide: a monomeric unit of DNA or RNA consisting of a sugarmoiety (pentose), a phosphate, and a nitrogenous heterocyclic base. Thebase is linked to the sugar moiety via the glycosidic carbon (1′ carbonof the pentose) and that combination of base and sugar is a nucleoside.A nucleoside containing at least one phosphate group bonded to the 3′ or5′ position of the pentose is a nucleotide.

[0052] Base Pair (bp): a partnership of adenine (A) with thymine (T), orof cytosine (C) with guanine (G) in a double stranded DNA molecule. InRNA, uracil (U) is substituted for thymine. Generally the partnership isachieved through hydrogen bonding.

[0053] Nucleic Acid: a polymer of nucleotides, either single or doublestranded.

[0054] Gene: a nucleic acid whose nucleotide sequence codes for an RNAor a polypeptide. A gene can be either RNA or DNA.

[0055] cDNA: a single stranded DNA that is homologous to an mRNAsequence and does not contain any intronic sequences.

[0056] Sense: a nucleic acid molecule in the same sequence order andcomposition as the homolog mRNA. The sense conformation is indicatedwith a “+”, “s” or “sense” symbol.

[0057] Antisense: a nucleic acid molecule complementary to therespective mRNA molecule. The antisense conformation is indicated as a“−” symbol or with a “a” or “antisense” in front of the DNA or RNA,e.g., “aDNA” or “aRNA”.

[0058] Template: a nucleic acid molecule being copied by a nucleic acidpolymerase. A template can be single-stranded, double-stranded orpartially double-stranded, depending on the polymerase. The synthesizedcopy is complementary to the template, or to at least one strand of adouble-stranded or partially double-stranded template. Both RNA and DNAare synthesized in the 5′ to 3′ direction. The two strands of a nucleicacid duplex are always aligned so that the 5′ ends of the two strandsare at opposite ends of the duplex (and, by necessity, so then are the3′ ends).

[0059] Nucleic Acid Template: a double-stranded DNA molecule, doublestranded RNA molecule, hybrid molecules such as DNA-RNA or RNA-DNAhybrid, or single-stranded DNA or RNA molecule.

[0060] Oligonucleotide: a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three, andusually more than ten. The exact size will depend on many factors, whichin turn depends on the ultimate function or use of the oligonucleotide.The oligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, or a combinationthereof.

[0061] Primer: an oligonucleotide complementary to a template. Theprimer complexes with the template to yield a primer/template duplex forinitiation of synthesis by a DNA polymerase. The primer/template complexis extended during DNA synthesis by the addition of covalently bondedbases linked at the 3′ end, which are complementary to the template. Theresult is a primer extension product. Virtually all known DNApolymerases (including reverse transcriptases) require complexing of anoligonucleotide to a single-stranded template (“priming”) to initiateDNA synthesis. A primer is selected to be “substantially” or“sufficiently” complementary to a strand of specific sequence of thetemplate. A primer must be sufficiently complementary to hybridize witha template strand for primer elongation to occur. A primer sequence neednot reflect the exact sequence of the template. For example, anon-complementary nucleotide fragment may be attached to the 5′ end ofthe primer, with the remainder of the primer sequence beingsubstantially complementary to the strand. Non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer sequence has sufficient complementarity with the sequence of thetemplate to hybridize and thereby form a template/primer complex forsynthesis of the extension product of the primer.

[0062] Complementary or Complementarity or Complementation: used inreference to polynucleotides (i.e., a sequence of nucleotides) relatedby the base-pairing rules. For example, the sequence “A-G-T” iscomplementary to the sequence “T-C-A,” and also to “T-C-U.”Complementation can be between two DNA strands, a DNA and an RNA strand,or between two RNA strands. Complementarity may be “partial” or“complete” or “total”. Partial complementarity or complementation occurswhen only some of the nucleic acid bases are matched according to thebase pairing rules. Complete or total complementarity or complementationoccurs when the bases are completely matched between the nucleic acidstrands. The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This is of particular importance inamplification reactions, as well as in detection methods that depend onbinding between nucleic acids. Percent complementarity orcomplementation refers to the number of mismatch bases over the totalbases in one strand of the nucleic acid. Thus, a 50% complementationmeans that half of the bases were mismatched and half were matched. Twostrands of nucleic acid can be complementary even though the two strandsdiffer in the number of bases. In this situation, the complementationoccurs between the portion of the longer strand corresponding to thebases on that strand that pair with the bases on the shorter strand.

[0063] Homologous or homology: refers to a polynucleotide sequencehaving similarities with a gene or mRNA sequence. A nucleic acidsequence may be partially or completely homologous to a particular geneor mRNA sequence, for example. Homology may also be expressed as apercentage determined by the number of similar nucleotides over thetotal number of nucleotides.

[0064] Complementary Bases: nucleotides that normally pair up when DNAor RNA adopts a double stranded configuration.

[0065] Complementary Nucleotide Sequence: a sequence of nucleotides in asingle-stranded molecule of DNA or RNA that is sufficientlycomplementary to that on another single strand to specifically hybridizebetween the two strands with consequent hydrogen bonding.

[0066] Conserved: a nucleotide sequence is conserved with respect to apreselected (reference) sequence if it non-randomly hybridizes to anexact or total complement of the preselected sequence.

[0067] Hybridize and Hybridization: the formation of complexes betweennucleotide sequences which are sufficiently complementary to formcomplexes via complementary base pairing. Where a primer (or splicetemplate) “hybridizes” with target (template), such complexes (orhybrids) are sufficiently stable to serve the priming function requiredby a DNA polymerase to initiate DNA synthesis. There is a specific, i.e.non-random, interaction between two complementary polynucleotide thatcan be competitively inhibited.

[0068] Nucleotide Analog: a purine or pyrimidine nucleotide that differsstructurally from T, G, C, or U, but is sufficiently similar tosubstitute for the normal nucleotide in a nucleic acid molecule.

[0069] DNA Homolog: a nucleic acid having a preselected conservednucleotide sequence and a sequence coding for a receptor capable ofbinding a preselected ligand.

[0070] Promoter-Linked Primer: an RNA-polymerase-promoter sense sequencecoupled with a gene-specific complementary sequence in its 3′ portionfor annealing to the antisense conformation of a nucleic acid template.

[0071] Amplification: nucleic acid replication involving templatespecificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in that they aresought to be sorted out from other nucleic acids. Amplificationtechniques have been designed primarily for this sorting. Templatespecificity is achieved in most amplification techniques by the choiceof enzyme. Amplification enzymes are enzymes that will process onlyspecific sequences of nucleic acid in a heterogeneous mixture of nucleicacids. For example, in the case of Qβ replicase, MDV-1 RNA is thespecific template for the replicase (Kacian et al. (1972) Proc. Natl.Acad. Sci. USA 69, 3038). Other nucleic acid will not be replicated bythis amplification enzyme. Similarly, T7 RNA polymerase has a stringentspecificity for its own promoters (Chamberlin et al. (1970) Nature 228,227). Taq and Pfu polymerases, by virtue of their ability to function athigh temperature, display high specificity for the sequences bonded, andthus defined by the primers.

[0072] Enzymatic Amplification: (such as PCR, NASBA and RNA-PCR): amethod for increasing the concentration of a segment in a targetsequence from a mixture of nucleic acids without cloning or purification(U.S. Pat. Nos. 4,683,195; 4,683,202; 4,965,188 (PCR); U.S. Pat. No.5,888,779 (NASBA); U.S. Pat. No. 6,197,554 (RNA-PCR) and WO 00/75356,hereby incorporated by reference). Amplification of the target sequenceby PCR consists of introducing a large excess of two oligonucleotideprimers to the DNA mixture containing the desired target sequence,followed by a precise sequence of thermal cycling in the presence of DNAand/or RNA polymerase(s). The two primers are complementary to theirrespective strands of the double stranded target sequence. To effectamplification, the mixture is denatured and the primers then annealed tocomplementary sequences within the target molecule. Following annealing,the primers are extended with a polymerase so as to form a new pair ofcomplementary strands. The steps of denaturation, primer annealing andpolymerase extension can be repeated many times (i.e., denaturation,annealing and extension constitute one “cycle”; there can be numerous“cycles”) to obtain a high concentration of an amplified segment of thedesired target sequence. The length of the amplified segment of thedesired target sequence is determined by the relative positions of theprimers with respect to each other, and therefore, this length is acontrollable parameter. Because the desired amplified segments of thetarget sequence become the predominant sequences (in terms ofconcentration) in the mixture, they are said to be amplified. Withenzymatic amplification, it is possible to amplify a single copy of aspecific target sequence in genomic DNA to a level detectable by severaldifferent methodologies (e.g., incorporation of biotinylated primersfollowed by avidin-enzyme conjugate detection; incorporation of ³²Plabeled triphosphates, such as dCTP or dATP, into the amplifiedsegment). In addition to genomic DNA, any oligonucleotide orpolynucleotide sequence can be amplified with the appropriate set ofprimer molecules. In particular, the amplified segments created by thePCR and RNA-PCR process itself are, themselves, efficient templates forsubsequent PCR and RNA-PCR amplification.

[0073] Polymerase Chain Reaction (PCR): an amplification reaction istypically carried out by cycling i.e., simultaneously performing in oneadmixture, the first and second primer extension reactions, each cyclecomprising polynucleotide synthesis followed by denaturation of thedouble stranded polynucleotides formed. Methods and systems foramplifying a DNA homolog are described in U.S. Pat. Nos. 4,683,195 and4,683,202, both to Mullis et al.

[0074] Amplifiable Nucleic Acid and Amplified Products: nucleic acidsthat may be amplified by any amplification method.

[0075] DNA-dependent DNA Polymerase: an enzyme that synthesizes acomplementary DNA copy from a DNA template. Examples are DNA polymeraseI from E. coli and bacteriophage T7 DNA polymerase. Under suitableconditions a DNA-dependent DNA polymerase may synthesize a complementaryDNA copy from an RNA template.

[0076] DNA-dependent RNA Polymerase or Transcriptase: enzymes thatsynthesize multiple RNA copies from a double stranded or partiallydouble stranded DNA molecule having a promoter sequence. Examples oftranscriptases include, but are not limited to, DNA-dependent RNApolymerase from E. coli and bacteriophage T7, T3, and SP6.

[0077] RNA-dependent DNA Polymerase or Reverse Transcriptase: enzymesthat synthesize a complementary DNA copy from an RNA template. All knownreverse transcriptases also have the ability to make a complementary DNAcopy from a DNA template. Thus, reverse transcriptases are bothRNA-dependent and DNA-dependent DNA polymerases.

[0078] RNase H: an enzyme that degrades the RNA portion of an RNA/DNAduplex. RNase H may be an endonuclease or an exonuclease. Most reversetranscriptase enzymes normally contain an RNase H activity. However,other sources of RNase H are available, without an associated polymeraseactivity. The degradation may result in separation of the RNA from aRNA/DNA complex. Alternatively, the RNase H may simply cut the RNA atvarious locations such that pieces of the RNA melt off or aresusceptible to enzymes that unwind portions of the RNA.

[0079] Reverse Transcription: the synthesis of a DNA molecule from anRNA molecule using an enzymatic reaction in vitro. For example, the RNAmolecule may be primed with a primer that is complementary to the RNAmolecule and the DNA molecule is synthesized by extension using areverse transcriptase such as Tth DNA polymerase with reversetranscription activity, MMLV reverse transcriptase, AMV reversetranscriptase, and any other enzyme that has the ability to synthesize aDNA molecule from an RNA molecule template.

[0080] In Vitro Transcription: the synthesis of an RNA molecule from aDNA molecule using an enzymatic reaction in vitro. For example, the DNAmolecule may be double stranded and comprises an RNA polymerase promotersuch as T7, SP6, T3, or any other enzyme promoter for synthesis of RNAfrom DNA.

[0081] Vector: a recombinant nucleic acid molecule such as recombinantDNA (rDNA) capable of movement and residence in different geneticenvironments. Generally, another nucleic acid is operatively linkedtherein. The vector can be capable of autonomous replication in a cellin which case the vector and the attached segment is replicated. Onetype of preferred vector is an episome, i.e., a nucleic acid moleculecapable of extrachromosomal replication. Preferred vectors are thosecapable of autonomous replication and/or expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes encoding for one or more polypeptides are referred to herein as“expression vectors”. Particularly important vectors allow cloning ofcDNA from mRNAs produced using a reverse transcriptase.

[0082] In Vitro Transcrintion/Translation Unit: a polynucleotidesequence comprising all the necessary nucleic acid sequence elements foroperably linking a desired nucleic acid sequence and regulatory elementsfor in vitro transcription and optionally translation reactions forexpressing the operably inserted nucleic acid sequence into an mRNA andoptionally a polypeptide in an in vitro system without a host cell.

[0083] Functional parts: a portion of an intact molecule that retainsone or more desired properties of the intact molecules. Thus, forexample, an antibody binds an antigen. In that context of the propertyof binding that antigen, a functional part of an antibody can be anyportion of an antibody that binds the cognate antigen. Similarly, afunctional part of a nucleic acid that encodes an antibody that bindsthat antigen is any portion of that nucleic acid that encodes apolypeptide that binds to that antigen.

[0084] Receptor: a molecule, such as a protein, glycoprotein and thelike, that can specifically (non-randomly) bind to another molecule. Anantibody is an example of a receptor.

[0085] Antibody: in various grammatical forms as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain a combining sitefor antigen or paratope. Exemplary antibody molecules are intactimmunoglobulin molecules, substantially intact immunoglobulin moleculesand portions of an immunoglobulin molecules, including those portionsknown in the art as F_(ab), F_(ab′), (F_(ab′))₂, F_(v) and scF_(v).

[0086] Antibody Combining Site: an antibody combining site is thatstructural portion of an antibody molecule comprised of a heavy andlight chain variable and hypervariable regions that specifically binds(immunoreacts with) an antigen.

[0087] Immunoreact: in various forms means specific binding between anantigenic determinant-containing molecule and a molecule containing anantibody combining site such as a whole antibody molecule or a portionthereof.

[0088] Fusion Polypeptide: a polypeptide comprised of at least twopolypeptides and optionally a linking sequence to operatively link thetwo polypeptides into one continuous polypeptide. The two polypeptideslinked in a fusion polypeptide are typically derived from twoindependent sources, and therefore a fusion polypeptide comprises twolinked polypeptides not normally found linked in nature.

[0089] Cistron: a sequence of nucleotides in a DNA molecule coding foran amino acid residue sequence and including upstream and downstream DNAexpression control elements.

[0090] Promoter: a nucleic acid to which a polymerase moleculerecognizes, perhaps binds to, and initiates synthesis. For the purposesof the instant invention, a promoter can be a known polymerase bindingsite, an enhancer and the like, any sequence that can initiate synthesisby a desired polymerase.

[0091] Replicase: an RNA-dependent RNA polymerase such as Brome mosaicvirus replicase, togaviridae virus replicase, Flock house virusreplicase and Qβ replicase.

[0092] Rearranged B cells: B cells in which immunoglobulin genetranslocation, i.e., rearrangement, has occurred as evidenced by thepresence in the cell of mRNA with the immunoglobulin gene V, D and Jregion transcripts adjacently located thereon.

[0093] B. Methods

[0094] The present invention provides a novel method for amplifying andcloning the variable regions of the immunoglobulin heavy (V_(H)) andlight (V_(L) of V_(K) and V_(λ)) chains to generate a very diverse humanantibody library representing the vast immunological repertoire. TheV_(H) and V_(L) DNA homologs are further expressed in either separateforms for an antibody receptor heterodimeric molecule or as a fusionsuch as an scF_(v) antibody, capable of binding a preselected ligand.Distinctively different from the PCR-based amplification method (U.S.Pat. No. 6,291,158 to Winter, et al.), the present invention generallyuses non-PCR-based amplification methods, in specific, atranscription-based amplification (TCA) for amplifying the V_(H) andV_(L) DNA homologs. The DNA homologs of the amplified V_(H) and V_(L)are further cloned into framework expression vectors known in the art.Heterodimeric antibody receptor or single chain scF_(v) antibodies areexpressed either in a host cell or in an in vitro transcription andtranslation (TnT) system. The present invention combines the followingelements as discussed in details thereafter:

[0095] 1. isolation of nucleic acids (genomic DNAs or mRNAs) fromimmunological tissue or cell sources containing a substantial portion ofthe immunological repertoire;

[0096] 2. preparation of oligonucleotide primers and promoter-linkedprimers for amplifying and cloning DNA homologs containingimmunoglobulin V_(H) and V_(L) variable regions of the immunoglobulinheavy and light chain genes;

[0097] 3. amplification and cloning of a gene library containing asubstantial population of different V_(H) and V_(L) genes;

[0098] 4a. in vivo expression of the V_(H) and V_(L) polypeptides in anappropriate host, including prokaryotic and eukaryotic hosts, eitherseparately or in the same cell, either on the same or differentexpression vectors, and either in linked single chain (scF_(v)) form orseparate heterodimeric receptor form; or alternatively, 4b. in vitroexpression of the V_(H) and V_(L) polypeptides in an in vitrotranscription and/or translation system, either in the same or differentframework, and either in linked single chain (scF_(v)) form or inseparate heterodimeric receptor form; and

[0099] 5. screening the antibody library for antibodies with apreselected activity.

[0100] As generally known in the art, the composition and length of theV_(H) and V_(L) vary widely, depending on the particular idiotypeinvolved. Typically, the individual V_(H) and V_(L) polypeptides havefewer than 125 amino acid residues, more usually between 60 to 120 aminoacid residues. The V_(H) polypeptides are often 110 to 125 amino acidresidues in length while V_(L) polypeptides are 95 to 115 amino acidresidues in length. Usually, there are at least two cysteines separatedby from about 60 to 75 amino acid residues and are joined by a disulfidebond. The V_(H) and V_(L) polypeptides produced by the subject inventionwill normally be substantial faithful copies of idiotypes of thevariable regions of the heavy and/or light chains of immunoglobulins,however, these polypeptides can be further mutated by site-specific orrandom mutagenesis to advantageously improve the desiredantigen-combining specificity and affinity. Typically the C terminusregion of the V_(H) and V_(L) polypeptides would have a greater varietyof the sequences than would the N terminus and, based on the presentinvention, can be further modified through either site directed orrandom mutagenesis to generate greater diversity than the normallyoccurring V_(H) and V_(L) polypeptides. A synthetic oligonucleotide canbe employed to vary one or more amino acids in a hypervariable region ofthe V_(H) and/or V_(L) polypeptides.

[0101] A V_(H) or V_(L) polypeptide can be produced separately as twoseparate transcripts from a single or two different vectors by thesubject invention and can be active in monomeric as well as multimericforms, either homomeric or heteromeric, preferably heterodimeric. V_(H)and V_(L) polypeptides produced by the present invention can beadvantageously combined in a heterodimeric antibody to offer uniqueantigen combining activities.

[0102] The present invention can produce also the F_(ab) antibody as aheterodimer comprised of a V_(H) polypeptide tagged with a portion ofthe heavy chain constant region and a V_(L) polypeptide tagged withsubstantially all of the light chain constant region. The production ofF_(ab) can be advantageous in some situations because the additionalconstant region sequences contained in a F_(ab) as compared to a F_(v)could stabilize the V_(H) and V_(L) interactive conformation. Suchstabilization may potentially increase the affinity of the F_(ab) forcorresponding antigen. In addition, the F_(ab) is more commonly used inthe art and thus there are more commercial antibodies available tospecifically recognize an F_(ab), especially to the constant regions ofan F_(ab).

[0103] Preferably the antibody produced by the subject invention issingle chain or the scF_(v) and is therefore normally comprised of theV_(H) and V_(L) linked with an artificial linker such as G₄S orGlyGlyGlyGlySer linker peptide, or multiple copies of such a linker,such as (G₄S)_(n). The V_(H) and V_(L) portions bend together to assumea conformation having a binding affinity, or association constant forthe preselected antibody that is different, preferably higher, than theaffinity or association constant of either of the V_(H) or V_(L)polypeptides alone, i.e. as monomers. This single-chain antigen-bindingantibody has been described by Bird et al., Science, 242:423-426 (1988).The design of suitable peptide linker regions is described in U.S. Pat.No. 4,704,692 by Robert Ladner. Such a peptide linker could be designedas part of the nucleic acid sequences contained in the expression vectoror in the in vitro translation framework sequences. The nucleic acidsequences coding for the peptide linker would be between the V_(H) andV_(L) DNA homologs and the restriction endonuclease sites used tooperatively link the V_(H) and V_(L) DNA homologs to the expressionvector. Such a peptide linker could also be coded by nucleic acidsequences that are part of the oligonucleotide primers used to preparethe V_(H) and V_(L) gene libraries so that overlapping sequences can befused to form the scF_(v) DNA homologs for cloning into an appropriateexpression vector or framework sequences.

[0104] An antibody produced by the present invention possesses aspecific combining activity and conformation having a binding sitespecific for an antigen as evidenced by its ability to be competitivelyinhibited. In one embodiment, an antibody of this invention possesses anantigen-combining binding site and can be selected by the ability tospecifically bind to a preselected antigen to form an immunoreactionproduct (complex) with a preselected antigen having a sufficientlystrong binding between the antigen and the binding site for theimmunoreaction product to be isolated. The antibody typically has anaffinity or avidity that is generally greater than 10⁵M⁻¹, more usuallygreater than 10⁶, and preferably greater than 10⁸M⁻¹.

[0105] In another embodiment, an antibody produced by the subjectinvention possesses catalytic activities by binding to a substrate andcatalyzing the formation of a product from the substrate. The topologyor conformation of the ligand-combining site of a catalytic antibody isprobably more important for the preselected catalytic activity than theaffinity (association constant or pK_(a)) for the substrate. The subjectcatalytic antibodies are preferred to have an association constant forthe preselected substrate generally greater than 10³M⁻¹, more usuallygreater than 10⁴M⁻¹ or 10⁵M⁻¹, and preferably greater than 10⁷M⁻¹.

[0106] 1. Isolation of nucleic acids (genomic DNAs or mRNAs) fromimmunological tissue or cell sources containing a substantial portion ofthe immunological repertoire:

[0107] As a general rule, the preferred starting source materials ortissues (i.e. peripheral blood, bone marrow, spleen or regional lymphnodes) for the antibody repertoire include, but are not limited to, aheterogeneous population of antibody producing cells, i.e. B lymphocytes(B cells), preferably rearranged B cells such as those found in thecirculation or spleen of a vertebrate. It is generally known in the artthat the greater the genetic heterogeneity of the population of cellsfor which the source nucleic acids are obtained, the greater thediversity of the immunological repertoire that will be made availablefor screening. Preferably, blood cells from different individuals,particularly those having an immunologically significant age difference,and different races or species can be advantageously combined to1ncrease the diversity of the repertoire.

[0108] In certain cases, it is desirable to enrich the immunoglobulingene repertoire for antibodies with higher affinity to a preselectedactivity, such as by using as a source of blood cells (source cells)from cancer patients, patients with autoimmune diseases or people in anyone of various stages of age, health and immune response or from animalsthrough repeated immunization.

[0109] In one preferred embodiment, the source cells are obtained frompooled human blood cells of cancer patients with high affinityantibodies against specific cancer or cancers. In another embodiment,the source cells are obtained from pooled human blood cells of patientswith autoimmune diseases such as rheumatoid arthritis, psoriasis, etc.

[0110] Nucleic acids such as genomic DNAs or mRNAs coding for V_(H) andV_(L) polypeptides can be derived from cells producing IgA, IgD, IgE,IgG or IgM, most preferably from IgM-producing cells and IgG-producingcells. The desired V_(H) and V_(L) gene repertoire can be isolated fromeither genomic DNA or the messenger RNA (mRNA) containing transcripts ofthe variable regions. It may be less desirable to use the genomic DNAfrom non-rearranged B lymphocytes, wherein the sequences coding for thevariable region are juxtaposed or separated by intronic or interveningsequences. To be useful for making the V_(H) and/or V_(L) gene library,the DNA fragment(s) containing the proper exons of the variable regionsmust first be isolated, the introns excised, and the exons then splicedin the proper order and in the proper orientation. It is, however,relatively easier to use rearranged B cells as source materials forcloning the V_(H) and V_(L) regions because the C, D and Jimmunoglobulin gene regions have translocated to become adjacent andcontinuous (free of introns) for the entire variable regions.

[0111] Methods for preparing fragments of genomic DNA from whichimmunoglobulin heavy and light chain variable region genes can beamplified and cloned are well known in the art, see for example Herrmannet al., Methods In Enzymol., 152:180-183, (1987); Frischauf, Methods InEnzymol., 152:183-190 (1987); Frischauf, Methods In Enzymol.,152:190-199 (1987); and DiLella et al., Methods In Enzymol., 152:199-212(1987).

[0112] Methods for isolating mRNA from source cells are known in theart. The procedure typically comprises lysis of cells under RNaseinhibiting conditions. In one embodiment, the total cellular mRNA isisolated by employing an oligo-dT cellulose column, see for exampleSambrook et al., “Molecular Cloning, 2^(nd) ed.”, Cold Spring HarborLaboratory Press.

[0113] 2. Preparation of oligonucleotide primers and promoter-linkedprimers for amplifying and cloning DNA homologs containingimmunoglobulin V_(H) and V_(L) (V_(K) and V_(λ)) variable regions of theimmunoglobulin heavy and light chain genes.

[0114] The oligonucleotide primers and the gene-specific primersequences of the promoter-linked primers used herein are selected to be“substantially” complementary to the different strands of each specificsequence to be synthesized or amplified. The primer so selected issufficiently complementary to nonrandomly hybridize with the respectivetemplate strand. In the case of the promoter-linked primers, anon-complementary RNA promoter sequence is attached to the 5′ end of thegene-specific primer, with the gene-specific primer sequence beingsubstantially complementary to the strand for nonrandom hybridization.The oligonucleotide primer and/or the promoter-linked primer can alsohave noncomplementary fragments for an endonuclease restriction site forcloning purposes.

[0115] The selection of a gene-specific primer for the V_(H) and V_(L)(V_(K) and V_(λ)) depends on various factors such as the distance on thenucleic acid from the region coding for the desired receptor, itshybridization site on the nucleic acid relative to any second primer tobe used, the number of genes in the repertoire it is to hybridize to,and the like.

[0116] For example, to produce V_(H) DNA homologs by the subjectinvention, the nucleotide sequence of a primer (including the primersegment of the promoter-linked primers) is selected to hybridize with aplurality of immunoglobulin heavy chain genes at a site substantiallyadjacent to the V_(H) region so that a nucleotide sequence coding for afunctional (capable of binding) polypeptide is obtained. To hybridize toa plurality of different V_(H) nucleic acid strands, the primer must bea substantial complement of a nucleotide sequence conserved among thedifferent strands. Such sites include nucleotide sequences in theconstant region, any of the variable region framework regions, the thirdframework region, leader region, promoter region, J region and the like.

[0117] In the present invention, the V_(H) and V_(L) DNA homologs areproduced by RNA transcription-based amplification. Two oligonucleotides,one gene-specific primer and one promoter-linked primer, which containsa gene specific primer and an RNA promoter sequence linked to the 5′-endof the gene-specific primer or the poly(dC)n oligonucleotide in the caseof using a terminal tailing reaction as depicted in FIG. 4, can be usedfor each strand of nucleic acid to be amplified. The RNA promotersequence can be linked to either the sense or the antisense primer to bethe promoter-linked primer for RNA transcription. Table 1, Table 2 andTable 3 list sets of gene-specific primers for both the gene-specificprimers and promoter-linked primers for V_(H), V_(K) and V_(λ) with theT7 RNA promoter sequence linked with either the sense or antisensegene-specific primers.

[0118] The oligonucleotide primers and the promoter-linked primers canbe prepared using any suitable method, such as, for example, thephosphotriester or phosphodiester methods see Narang et al., Meth.Enzymol., 68:90, (1979); U.S. Pat. No.4,356,270; and Brown et al., Meth.Enzymol., 68:109, (1979).

[0119] 3. Amplification and cloning of gene libraries containing asubstantial population of different V_(H) and/or V_(L) genes willdepend, as is well known in the art, on the type, complexity, and purityof the nucleic acids making up the input repertoire. Other factorsinclude whether or not the genes are to be amplified and/or mutagenized.The amplification and cloning strategies of the present invention aredependent also on the input repertoire of the double stranded genomicDNA or the mRNA isolated from the source immunological cells, preferablythe human blood cells.

[0120] In one embodiment, the isolated mRNAs of a tissue or cells ofimmunological origin are the source materials for the immunoglobulingene repertoire. As depicted in FIG. 1, the mRNAs of the immunoglobulingenes can be reversed transcribed using an antisense gene-specificprimer linked with the RNA promoter sequence for a DNA-dependent RNApolymerase such as the T7 promoter (the promoter-linked primer, P1). TheP1 primer is situated in the conserved region adjacent to the variabledomain such as in the J regions of the heavy or light chains. Theresulting cDNAs are made into double-stranded (ds) cDNAs by methodsknown in the art, such as using the RNA priming with RNase H treatmentas depicted in FIG. 1 or by priming with a sense gene-specific primer(P2) situated in the relatively conserved region of the V_(H) or V_(L)as depicted in FIG. 2. In both embodiments of FIGS. 2 and 3, the dscDNAs are then transcribed by the DNA-dependent RNA polymerase, such asT7 polymerase, into up to 2,000 copies of antisense RNA transcripts. Theamplified antisense RNA transcripts can be amplified again. Theantisense RNA transcripts are reverse transcribed into sense DNAs(sDNAs) by a reverse transcriptase using a sense P2 primer, thesingle-stranded sDNAs are made into ds DNAs with the P1 primer of thepromoter-linked primer. The resulting ds DNAs can be transcribed into upto 2,000 copies of antisense RNA transcripts again by the DNA-dependentRNA polymerase. With two cycles of transcription amplification, theoriginal mRNAs of the V_(H) or V_(L) gene repertoire are amplified orreproduced by hundreds of thousands to millions fold of antisense RNAtranscripts complementary to the original mRNA sequences. The aboveprocess can be repeated or cycled a couple of more times, if needed. Theresulting antisense RNA transcripts can be readily converted intoclonable ds DNAs by techniques known in the art and the resultingclonable ds V_(H) and V_(L) DNA homologs are ready for further cloningand/or expression in in vitro transcription and/or translation systemsor in an in vivo expression vector in a host cell.

[0121] In another embodiment, the V_(H) and V_(L) of the heavy and lightchain gene repertoire are amplified into sense RNA transcripts. In oneembodiment as depicted in FIG. 3, the antisense P1 primer is not linkedwith the RNA promoter sequence, rather, the sense P2 primer is linkedwith RNA promoter sequence. The mRNA transcripts are firstreversed-transcribed into cDNAs by the antisense P1 primer in thepresence of an antisense stop primer (the stop primer may or may nothave a terminator at the 3′-end) and reverse transcriptase. The stopprimer is situated immediately and adjacent to the 5′-terminus of thegene-specific portion of the sense P2 primer. The purpose of employing astop primer is to prevent the first strand cDNA primed by the antisenseP1 from over-extending beyond the P2 primer site so that ds cDNA can bemade using the antisense P2 primer linked with the RNA promoter sequence(the promoter-linked primer) to generate ds RNA promoter sequences forRNA transcription. The ds cDNAs are made by the sense P2 primerextension on the first single-stranded (ss) cDNAs and ds RNA promotertemplate is made in the simultaneous extension of the first ss cDNAswith the RNA promoter oligonucleotide sequence in the sense P2promoter-linked primer. The resulting ds cDNAs are transcribed intohundreds to thousands of copies of sense RNA transcripts. The sense RNAtranscripts are amplified by antisense P1 reverse priming in a reversetranscription reaction and double-stranding by the sense P2 primer andRNA transcription of the ds DNA homologs, which can be further amplifiedby repeating the same process a few more times to generate the clonableV_(H) and V_(L) DNA homologs.

[0122] In another embodiment as depicted in FIG. 4, the antisense P1primer is not linked with the RNA promoter sequence. The first strandcDNAs are synthesized by reverse priming of the mRNA by the antisense P1primer. The 3′-end of the resultant cDNAs is modified with a poly(G)_(n) tail by a terminal transferase (TdT). The sense P2 primer is apoly (C)_(n)GG oligonucleotide linked with an RNA promoter sequence atthe 5′-end. Double-stranded cDNAs are made by the sense P2 primerextension on the first strand cDNAs and the resulting ds cDNAs are usedas the templates for the RNA polymerase to transcribe hundreds tothousands of copies of sense RNA transcripts. The sense RNA transcriptscan be amplified further by firstly copied into cDNAs by reversetranscriptase from P1 primer and then tailed with poly (G)_(n). The sstailed cDNAs are primed with the sense P2 primer and converted into dscDNA again as templates for RNA transcription amplification. Thisprocess can be repeated a couple of more times for sufficientamplification of the RNA transcripts of the V_(H) and V_(L) regions ofthe immunological heavy and light chain genes. The resulting RNAtranscripts can be converted into ds DNAs by methods known in the art toproduce the desired V_(H) and V_(L) DNA homologs for downstream cloningand expression purposes. The sense RNA transcripts contain the 5′terminal portions of the mRNA transcripts including, for example,ribosome binding sites and translation initiation sites.

[0123] In another embodiment wherein the immunoglobulin repertoiresources are genomic DNAs from immunological tissues or cells, such as ahybridoma or stimulated immunological cells with rearrangedimmunoglobulin genes, the desired V_(H) and V_(L) DNA homologs are made,for example, as depicted in FIG. 5 or FIG. 6. The genomic DNAs areusually first denatured, typically by melting, into single strands. Inone embodiment as depicted in FIG. 5, the antisense P1 primer is linkedwith the RNA promoter sequence and used to copy only once the sense DNAtemplate into an antisense strand DNAs which are copied by the sense P2primer into ds DNA containing the ds RNA promoter template at the P1primer end for RNA transcription amplification. The amplified RNAtranscripts are in the antisense orientation and are reverse transcribedinto sense DNA by the sense P2 primer and reverse transcriptase. Thesense-oriented ss DNAs are further copied into ds DNA by the antisenseP1 primer and a DNA polymerase into the V_(H) and V_(L) DNA homologs,which can be further amplified by repeating the process. Thesufficiently amplified V_(H) and V_(L) DNA homologs are clonable in thedownstream cloning and expression exercise. In another embodiment asdepicted in FIG. 6, the ds genomic DNAs are first copied once intoantisense DNAs by the antisense P1 primer and a DNA-dependent DNApolymerase in the presence of a stop primer as described there before inFIG. 3. The copied antisense DNAs are further copied once by the RNApromoter-linked primer P2 into ds DNA and the ds RNA promoter templateis made by the extension of the first antisense DNAs stopped by the stopprimer at the adjacent site immediately 5′ to the end of the P2 primersite. The ds copied DNAs are subject to an RNA transcriptionamplification to generate sense RNA transcripts, which can be reversetranscribed into ss cDNAs by the antisense P1 primer and a reversetranscriptase. The ss cDNAs then are double stranded by the sense P2promoter-linked primer. The resulting ds DNAs can be further amplifiedby repeating the process to generate sufficient amounts of the V_(H) andV_(L) homologs.

[0124] The present invention also contemplates V_(H) and V_(L) DNAhomolog production via co-amplification (using two pairs of primers),and multiplex amplification (using up to about 8, 9, 10 or more primerpairs of P1 and P2 type primers). As in the before discussedembodiments, a plurality of the P1 and P2 primers can be used in eachtranscription-based amplification, or an individual pair of P1 and P2primers can be used. In any case, the amplification products of thetranscription-based amplifications using the same or differentcombinations of P1 and P2 primers can be combined to increase thediversity of the gene library.

[0125] The DNA polymerization, reverse transcription and RNAtranscription so discussed in the embodiments herein are performed usingany suitable methods known in the art. Generally they occur in bufferedaqueous solutions at preferred pH conditions with the P1 and/or P2primers admixed with the buffers containing the template strand. A largemolar excess of the P1 and/or P2 primers is advantageous or preferred toimprove the efficiency of the processes involved.

[0126] The deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTPare admixed in the reverse transcription and the DNA polymerizationreactions for making the DNA molecules. The ribonucleotide triphosphatesATP, CTP, GTP and TTP are also admixed in the DNA-Dependent RNAtranscription reaction for making the RNA molecules.

[0127] Suitable enzymes for DNA-dependent DNA polymerization include,for example, E. coli, DNA polymerase I, Klenow fragment of E. coli DNApolymerase I, T4 DNA polymerase, other available DNA polymerases andother enzymes such as heat-stable enzymes such as Taq-like DNApolymerases, TTh-like DNA polymerase, C. therm polymerase, andcombinations thereof. The suitable enzymes for the DNA-dependent RNApolymerization include RNA polymerases such as T3 RNA polymerase, T7 RNApolymerase, SP6 RNA polymerase, M13 RNA polymerase and viral replicase.

[0128] Generally, the synthesis will be initiated at the 3′ end of eachprimer and proceed in the 5′ direction along the template strand, untilsynthesis terminates, producing molecules of different lengths. Theremay be inducing agents, however, which initiate synthesis at the 5′ endand proceed in the above direction, using the same process as describedabove.

[0129] The V_(H) and V_(L) DNA homologs produced by the presenttranscription-based amplification are typically in double-stranded formand have contiguous or adjacent to each of their termini a nucleotidesequence defining an endonuclease restriction site. Digestion of theV_(H) and V_(L) DNA homologs having restriction sites at or near theirtermini with one or more appropriate endonucleases results in theproduction of homologs having cohesive termini of predeterminedspecificity for purpose of cloning into a vector or framework sequences.

[0130] The expression of the antigen-combining molecules of V_(H) andV_(L) genes can be achieved either via in vitro transcription andtranslation or by expression in a host cell through cloning into anexpression vector known in the art. For in vitro transcription andtranslation expression of the immunological genes, the P1 and P2 primersare so designed to fuse appropriately into an antibody frameworksequence or other scaffold sequences, which contains sequences necessaryfor adequate coupling of in vitro transcription and translationutilizing methods known in the art. The expression vectors can beselected to ensure expression in a wide range of suitable host cells orin selected host cells such as E. coli or mammalian cells. The V_(H) andV_(L) gene repertoire can be operatively fused with various surfaceproteins of a filamentous bacteriophage for displaying on the surface ofthe bacterial phage known in the art as phage display, see for example,Gao et al., supra.

[0131] The V_(H) and V_(L) DNA homologs can be separately cloned,expressed and combined into heterodimeric antibodies. Alternatively, theV_(H) and V_(L) genes can be operatively linked by a synthetic linkerpeptide to form a single chain F_(v) or scF_(v) as known in the art.Both heterodimeric antibodies including the F_(ab) antibodies and thescF_(v) antibodies can be displayed by phage display systems orgenerated in an in vitro translation system known in the art.

[0132] In preferred embodiments, the transcription-based amplificationprocess of the present invention is used not only to amplify the V_(H)and/or V_(L) DNA homologs of the immunological gene repertoire, but alsoto induce mutations within the library and thereby provide a libraryhaving a greater heterogeneity. Mutations can be deliberately introducedin the V_(H) and V_(L) DNA homologs by certain error-prone thermostableDNA-dependent DNA polymerases such as Taq-like polymerases known in theart. In other cases, mutations can be induced during the DNApolymerization or reverse transcription reactions by incorporating intothe reaction admixture nucleotide derivatives such as inosine, xanthine,hypoxanthine, and other labeled nucleotides, not normally found in thenucleic acids of the repertoire being amplified. During subsequent invivo or in vitro amplification reactions, the nucleotide derivativeswill be replaced with substitute nucleotides thereby inducing pointmutations in the V_(H) and V_(L) DNA homolog repertoire.

[0133] While the above discussion relates to the cloning of DNAsequences, as known in the art, RNA's can be cloned also. Thus, thetranscribed single stranded RNA molecules, for example, particularlythose produced, when two primers are used, with the polymerase promoteron the second of the two primers.

[0134] 4a. In vivo expression of the V_(H) and V_(L) polypeptides in anappropriate host, including prokaryotic and eukaryotic hosts, eitherseparately or in the same cell, either on the same or differentexpression vectors, and either in linked single chain (scF_(v)) form orseparate heterodimeric receptor form. Alternatively, the V_(H) and/orV_(L) DNA homolog repertoire produced by the present invention have bydesign restriction enzyme sites for cloning into a vector foramplification and/or expression in a host cell.

[0135] The various vectors suitable for replicating and expressing theV_(H) and/or V_(L) gene repertoire are available from many commercialvendors and are well known in the art. Those vectors include aprokaryotic expression vector such as plasmid vector containing aprokaryotic promoter capable of directing the expression (transcriptionand translation) of the V_(H) and/or V_(L) DNA homologs in a bacterialhost cell, such as E. coli transformed therewith. Typical of suchplasmid vectors are pUC8, pUC9, pBR322, pBR329, pPL, pKK223 and othervectors known in the art and available from commercial vendors such asBioRad Laboratories (Richmond, Calif.), Amersham Biosciences(Piscataway, N.J.), Invitrogen (Carlsbad, Calif.) and Stratagene (LaJolla, Calif.). Those expression vectors also include eukaryoticexpression vectors for expressing polypeptides in eukaryotic cells suchas yeast and mammalian cells. Many eukaryotic expression vectors, suchas pSVL, pCDNAneo, etc., are well known in the art and are availablefrom several commercial sources. As well known in the art, bothprokaryotic and eukaryotic expression vectors contain selectable drugresistant drug markers such as ampicillin or tetracycline resistant genemarkers for prokaryotic vectors and neomycin selection marker foreukaryotic expression vectors. In preferred embodiments, the expressionof the V_(H) and/or V_(L) gene repertoire in mammalian cells are carriedout by using retroviral expression vectors, which vector sequencesincluding the promoter sequences are derived from the long terminalrepeat (LTR) region of a retrovirus genome. There are many retroviralexpression vectors are available from commercial vendors such asStratagene (La Jolla, Calif.) and Invitrogen (Carlsbad, Calif.) and arewell known in the art.

[0136] In preferred embodiments, diverse heterodimeric antibodies areexpressed from randomly combining of the V_(H) and V_(L) DNA homologs ofthe diverse libraries in vitro for polycistronic expression fromindividual vectors. A library of vectors is generated with each vectorcapable of expressing, under the control of a single promoter, one V_(H)DNA homolog and one V_(L) DNA homolog, with these V_(H) and V_(L) DNAhomologs being randomly combined to produce the heterodimeric antibodyin a single host cell.

[0137] The heterodimeric antibody of one V_(H) and one V_(L) combinationcan also be produced from two distinctive expression vectors with twodifferent drug resistant selection markers. A cell selectively survivingtwo drugs contains at least one V_(H) and at least one V_(L) to form arandomly combined heterodimeric antibody. In one preferred embodiment,the linear double stranded lambda vectors such as Lambda Zap or itsderivative vectors from Stratagene (La Jolla, Calif.) are used and arewell known in the art.

[0138] As well known in the art, each of the vectors discussed in thepresent invention may comprise a ribosome binding site, a leadersequence, a polylinker sequence for restriction enzyme sites, a stopcodon, a selectable marker, or a peptide tag in certain cases.

[0139] The generation of the diverse heterodimeric antibodies or therandom combination of the V_(H) and V_(L) is accomplished by ligatingV_(H) DNA homologs into a first vector, typically at a restriction siteor sites within the polylinker sequence of the vector. Similarly, V_(L)DNA homologs are ligated into a second vector, thereby creating twodiverse populations of expression vectors. It does not matter which typeof DNA homolog, i.e., V_(H) or V_(L), is ligated to which vector, but itis preferred to have all V_(H) DNA homologs ligated to either the firstor second vector and all V_(L) DNA homologs ligated to the other of thefirst or second vector. The members of both populations are then cleavedwith an endonuclease at the shared restriction site, typically bydigesting both populations with the same enzyme. The resulting productis two diverse populations of restriction fragments where the members ofone have cohesive termini complementary to the cohesive termini of themembers of the other. The restriction fragments of the two populationsare randomly ligated to one another, i.e., a random, interpopulationligation is performed, to produce a diverse population of vectors eachhaving a V_(H) and V_(L) DNA homolog located in the same reading frameand under the control of the promoter of the second vector. Subsequentrecombinations can be achieved through cleavage at the sharedrestriction site, which is typically reformed on ligation of membersfrom the two populations, followed by subsequent religations. Thediverse heterodimeric antibodies or the F_(v) antibodies of randomlycombined V_(H) and V_(L) are produced in host cells by transforming thehost cells with the before-described recombined V_(H) and V_(L) generepertoire.

[0140] The host cell for replicating the vectors and expressing theV_(H) and/or V_(L) gene repertoire can be either prokaryotic oreukaryotic. Bacterial cells are preferred prokaryotic host cells andtypically are a strain of E. coli such as, for example, the E. colistrain DH5 available from Invitrogen (Carlsbad, Calif.). Preferredeukaryotic host cells include yeast and mammalian cells, preferablyvertebrate cells such as those from a mouse, rat, monkey or human cellline.

[0141] Transformation of appropriate cell hosts with a recombinant DNAmolecule of the present invention is carried out by such methods aselectroporation, lipofection, and other transfection agents known in theart and available from many vendors, see, for example, Maniatis et al.,Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982).

[0142] 4b. In vitro expression of the V_(H) and V_(L) polypeptides in anin vitro transcription and/or translation system, either on the same ordifferent frameworks, and either in linked single chain (scF_(v)) formor separate heterodimeric receptor form can be practiced using materialsand methods known in the art, such as the PROfusion system described inU.S. Pat. No. 6,214,553 to Szostak, et al. In general, the PROfusiontechnology consists of an in vitro or in situ transcription/translationprotocol that generates protein covalently linked to the 3′ end of thevery mRNA, i.e., an RNA-protein fusion. This is accomplished bysynthesis and in vitro or in situ translation of an mRNA molecule with apeptide acceptor attached to the 3′ end of the message. One preferredpeptide acceptor is puromycin, a nucleoside analog that adds to theC-terminus of a growing peptide chain and terminates translation. In onepreferred design, a DNA sequence is included between the end of themessage and the peptide acceptor which is designed to cause the ribosometo pause at the end of the open reading frame, providing additional timefor the peptide acceptor (for example, puromycin) to accept the nascentpeptide chain before hydrolysis of the peptidyl-tRNA linkage. Typically,the mRNA-protein fusion system comprises an in vitro expression unitcomprising a untranslated region containing an RNA polymerase bindingsequence, a ribosome binding sequence, and a translation initiationsignal. The in vitro transcription and translation unit is to fuse withthe desired polynucleotide sequence tagged with stop codon and possiblythe poly-A site for expressing fused the polypeptide in frame.

[0143] In a preferred embodiment employing such in vitro transcriptionand translation systems to express the V_(H) and/or V_(L) generepertoire, the V_(H) DNA homologs are fused to the 3′-end of the firstin vitro transcription and translation unit capable of expressing oneV_(H) DNA homolog and likewise, the V_(L) DNA homologs are fused into asecond transcription and translation unit. The first and secondtranscription and translation units are subjected to in vitrotranscription and translation in the PROfusion system to produce theV_(H) and V_(L) gene repertoire, separately. The separately expressedV_(H) and V_(L) are mixed to form randomly combined heterodimeric F_(v)antibodies in vitro.

[0144] In another preferred embodiment, the V_(H) and V_(L) DNA homologsare fused with a linker oligonucleotide coding for a linker peptide suchas GlyGlyGlyGlySer (Gly₄Ser). The single chain polypeptide ofV_(H)-(GlyGlyGlyGlySer)_(n)-V_(L) or V_(L)-(GlyGlyGlyGlySer)_(n)-V_(H)are fused in frame of the transcription and translation unit for invitro transcription and translation in the PROfusion system to producescF_(v) antibodies or scF_(v) antibody library.

[0145] The heterodimeric antibodies or the scF_(v) antibodies can alsobe made employing the ribosome display technology as described in thePCT patent application WO 09105058A1 by Glenn Kawasaki. Ribosome displayis a method for producing polypeptides, comprising: (a) constructing anin vitro expression unit comprising an untranslated region containing anRNA polymerase binding sequence, a ribosome binding sequence, and atranslation initiation signal, said expression unit being capable ofproducing mRNA; (b) attaching one or more semi-random nucleotidesequences to said expression unit; (c) transcribing or replicating thesequences associated with the expression-unit and semi-random nucleotidesequences to produce RNA; (d) translating said RNA to produce polysomesunder conditions sufficient to maintain said polysomes; (e) binding saidpolysomes to a substance of interest; (f) isolating said polysomes thatbind to said substance of interest; (g) disrupting said isolatedpolysomes to release mRNA; (h) recovering said mRNA; (i) constructingcDNA from said recovered mRNA; and (j) expressing said cDNA to producenovel polypeptides.

[0146] Similar to the embodiments employing the PROfusion method, theheterodimeric or scF_(v) antibody libraries can be produced by fusingthe V_(H) or V_(L) or the V_(H)-(GlyGlyGlyGlySer)_(n)-V_(L) or theV_(L)-(GlyGlyGlyGlySer)_(n)-V_(H) molecules with the in vitrotranscription/translation unit sequence.

[0147] 5. The antibody libraries produced by the present invention canbe screened for preselected antigen binding or catalytic activities. Inthe case of using antibody libraries expressed in a host cell, thepreferred screening assays are those where the binding of ligand by thereceptor produces a detectable signal, either directly or indirectly.Such signals include, for example, the production of a complex,formation of a catalytic reaction product, the release or uptake ofenergy, and the like. In preferred embodiments, the immunologicalmethods as well known in the art are employed, especially to performimmunochemical assays against a preselected epitope or a ligand.

[0148] 6. The present invention contemplates an antibody gene library,preferably produced by a transcription-based amplification method asdescribed herein, containing at least about 10³, preferably at leastabout 10⁵, more preferably at least about 10⁷, more preferably at leastabout 10⁸ and most preferably at least 10⁹ different V_(H) and/or V_(L)DNA homologs.

[0149] In preferred embodiments, a substantial portion of the V_(H)and/or V_(L) DNA homologs present in the antibody library areoperatively linked in a vector, preferably operatively linked forexpression by an expression vector or operatively fused with an in vitrotranscription/translation unit.

[0150] The present invention contemplates a host cell or cellstransformed therewith an antibody library containing the V_(H) and/orV_(L) DNA homologs. The present invention also contemplates a mediumsuitable for in vitro transcription/translation therewithin having theV_(H) and/or V_(L) DNA homologs fused with the in vitrotranscription/translation unit. The medium comprises water, bufferingsalts and the like and the transcription/translation unit fused with theV_(H) and/or V_(L) DNA homologs.

[0151] The libraries of the instant invention are well represented withclones from a wide range of molecules. The instant methods enablecapture of genes previously poorly amplified or cloned, or not cloned atall. Moreover, by modifying the captured genes, for example, when a DNAor an RNA, the diversity of the library can be enhanced beyond whatrepresents the naturally occurring repertoire. For example, ageneralized mutagenesis or site-directed mutagenesis can be conducted onthe nucleic acids to promote diversity of the members of the library.

[0152] The antibodies and antigen-binding fragments and constructsthereof, are human antibodies. Thus, the risk of generating a “serumsickness” reaction to xenogenic, non-human epitopes is minimized. Theantibodies can find use in any of the art-recognized uses for antibodyand antibody-type molecules. For example, an antibody obtained from aninstant library can be used as an affinity reagent to purify antigenfrom a mixture. An antibody of the instant invention can be used in anassay, whether in vitro or in vivo. The antibody can be used in director indirect assays, can be labeled and so on as known in the art. Thus,an antibody of interest can be used in known diagnostic assays, such asfluorescence assays, ELISA's, RIA's and the like. As indicted, as ahuman antibody, the instant antibody is less antigenic and can be usedas, for example, an imaging agent along with an appropriate detectingdevice, such as a fluoroscope or a gamma camera. An instant antibodyalso can find use as a therapeutic agent. For example, the antibody canbe effective alone in disrupting a pathogen or pathogenic state in ahuman. Also, the antibody can be conjugated to a cytotoxic agent, suchas a radionuclide, a poison, such as ricin, and so on. Thus, theantibody serves as a targeting agent. There are many uses of antibodies,as known in the art, and any one of those uses is contemplated to bepracticable using an antibody obtained by the methods and from thelibraries of interest. The various methods of using an antibody are wellknown to the artisan, and such use is a design choice. Any of a numberof treatises can be consulted regarding the uses of antibody, andparticularly human antibody.

[0153] The invention now will be exemplified further in the followingnon-limiting examples.

EXAMPLES Example 1

[0154] Gene-Specific Oligonucleotide and RNA Promoter-Linked PrimerSelection

[0155] The nucleotide sequences coding for the human immunoglobulincomplimentary determining region (CDR) are highly variable (Marks, J. D.et al., J. Mol. Biol. 1991, 222, 581-597; Haidaris, C. G. et al. 2001,257, 185-202; Welschof, M., et al. J. Immunol. Methods 1995, 179,203-214.; Marks, J. D. et al. Eur. J. Immunol. 1991, 21, 985-991; andHaard, H. J. D. et al. J. Biol. Chem. 1999, 274, 18218-18230). However,there are several regions of conserved sequences that flank the humanV_(H) domains, containing substantially conserved nucleotide sequences,i.e., sequences that will hybridize to the same primer sequence in anumber of different genes. Therefore, gene-specific oligonucleotideprimers can be selected for both gene-specific primers and thepromoter-linked primers and synthesized to hybridize to the conservedsequences for reverse transcription, double stranding and RNAtranscription reactions as described in the present invention. Fortranscriptional amplification of the human V_(H) domains, theV_(H)-specific oligonucleotide primer sequences are either in the senseorientation or antisense orientation. In all cases, the sense primers(Table 1) were chosen to be either in the conserved N-terminus region ofthe human V_(H) domains and to be homologous to the sense mRNAtranscripts or complementary to the first strand cDNAs or at the 5′terminus, and the antisense primers were chosen to be in the flanking Jregion and to be complementary to the sense mRNA transcripts. To reducethe number of oligonucleotide primers to be synthesized, certain wobblenucleotides were incorporated into the gene-specific primer sequences.As known well in the art, the standard code letters for specifying awobble are: R=A/G, Y=C/T, M=A/C, K=G/T, S=C/G, W=A/T, B=C/G/T, D=A/G/T,H=A/C/T, V=A/C/G, and N=A/C/G/T. To amplify the V_(H) domains byproducing the antisense RNA transcripts intermediates as discussed inFIG. 1 and FIG. 2, the T7 RNA promoter sequence (T7:5′-dCCA GTG AAT TGTAAT ACG ACT CAC TAT AGG GAA-3′ (SEQ ID NO:__) is linked to the 5′ end ofthe antisense primers. Alternatively, the T7 RNA promoter sequence islinked to 5′ end of the sense primers to produce the sense RNAtranscripts intermediates as presented in FIG. 3 and FIG. 4.

[0156] Additionally, V_(H)-specific amplification includes uniqueantisense primers that were designed to be complementary to a portion ofthe first constant region domain of the γ₁ heavy chain mRNA. Theseprimers will produce V_(H) DNA homologs containing polynucleotidescoding for amino acids from the V_(H) and the first constant regiondomains of the heavy chain. These DNA homologs can therefore be used toproduce F_(ab) fragments rather than an F_(V). The primers may containrestriction sites, stop codons, peptide linkers and the like, asdisclosed herein. Restriction sites, stop codons and sequences encodinglinkers are underlined. TABLE 1 Human V_(H)-Specific Primers AntisenseEcoRI & Stop Codon-Linked HJ_(H) Primers: aHJ_(H)-1: 5′-dTGG AAT GAA TTCGAT TGC TAG TCA GAC GGT GAC CAG (SEQ ID NO:_)           GGT GCC-3′aHJ_(H)-2: 5′-dTGG AAT GAA TTC GAT TGC TAG TCA GAC GGT GAC CAT (SEQ IDNO:_)           TGT CCC-3′ aHJ_(H)-3: 5′-dTGG AAT GAA TTC GAT TGC TAGTCA GAC GGT GAC CAG (SEQ ID NO:_)           GGT TCC-3′ aHJ_(H)-4:5′-dTGG AAT GAA TTC GAT TGC TAG TCA GAC GGT GAC CGT (SEQ ID NO:_)          GGT CCC-3′ Sense T7 & Not I-Linked Primer: 5′-dCCA GTG AAT TGTAAT ACG ACT CAC TAT AGG GAA CGG CAT (SEQ ID NO:_)     GGAATG CGG CCG CCC CCC CCC C-3′. T7 & Peptide Linker(PL)-Linked AntisenseHJ_(H) Primers: aT7PLHJ_(H)-1:5′-dCCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GAA (SEQ ID NO_)               AGA GCC GCC GCC GCC TGA GGA GAC GGT GAC CAG               GGT GCC-3′ aT7PLHJ_(H)-2:5′-dCCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GAA (SEQ ID NO_)               AGA GCC GCC GCC GCC TGA GGA GAC GGT GAC CAT               TGT CCC-3′ aT7PLHJ_(H)-3:5′-dCCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GAA (SEQ ID NO_)               AGA GCC GCC GCC GCC TGA GGA GAC GGT GAC CAG               GGT TCC-3′ aT7PLHJ_(H)-4:5′-dCCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GAA (SEQ ID NO_)               AGA GCC GCC GCC GCC TGA GGA GAC GGT GAC CGT               GGT CCC-3′ Sense HV_(H) Primers: sHV_(H)-1: 5′-CAG CCGGCC ATG GCA CAG GTN CAG CTG GTR CAG TCT GG-3′ (SEQ ID NO:_) sHV_(H)-2:5′-CAG CCG GCC ATG GCA CAG GTC CAG CTG GTR CAG TCT GGG G-3′ (SEQ IDNO:_) sHV_(H)-3: 5′-CAG CCG GCC ATG GCA CAG GTK CAG CTG GTG SAG TGTGGG-3′ (SEQ ID NO:_) sHV_(H)-4: 5′-CAG CCG GCC ATG GCA CAG GTC ACC TTGARG GAG TCT GGT CC-3′ (SEQ ID NO:_) sHV_(H)-5: 5′-CAG CCG GCC ATG GCACAG GTG CAG CTG GTG GAG WCT GG-3′ (SEQ ID NO:_) sHV_(H)-6: 5′-CAG CCGGCC ATG GCA CAG GTG CAG CTG GTG SAG TCY GG-3′ (SEQ ID NO:_) sHV_(H)-7:5′-CAG CCG GCC ATG GCA CAG GTG CAG CTG CAG GAG TCG G-3′ (SEQ ID NO:_)sHV_(H)-8: 5′-CAG CCG GCC ATG GCA CAG GTG CAG CTG TTG SAG TCT G-3′ (SEQID NO:_) sHV_(H)-9: 5′-CAG CCG GCC ATG GCA CAG GTG CAG CTG GTG CAA TCTG-3′ (SEQ ID NO:_) sHV_(H)-10: 5′-CAG CCG GCC ATG GCA CAG GTG CAG CTGCAG GAG TCC GG-3′ (SEQ ID NO:_) sHV_(H)-11: 5′-CAG CCG GCC ATG GCA CAGGTG CAG CTA CAG CAG TGG G-3′ (SEQ ID NO:_) sHV_(H)-12: 5′-CAG CCG GCCATG GCA CAG GTA CAG CTG CAG CAG TCA G-3′ (SEQ ID NO:_)

[0157] The nucleotide sequences coding for the human V_(L) (both theV_(K) and V_(λ) isotypes) CDRs are also highly variable. However, thereare several regions of conserved sequences that flank the V_(L) CDRdomains including the J_(L), V_(L) framework regions and V_(L)leader/promoter. Therefore, VL-specific primers that hybridize to theconserved sequences are selected and synthesized in the similar fashionas for the human V_(H) domains as discussed herebefore.

[0158] Table 2 lists the human V_(K)-specific primers and Table 3 listshuman V_(λ)-specific primers used for the present invention. TABLE 2Human V_(K)-Specific Primers Antisense XhoI & Stop Codon-Linked HJ_(k)Primers: aHJ_(K)-1: 5′-dTGG AAT TCT CGA GAT TGC TAG TCA ACG TTT GAT TTCCAC (SEQ ID NO:_)             CTT GGT CCC-3′ aHJ_(K)-2: 5′-dTGG AATTCT CGA GAT TGC TAG TCA ACT TTT GAT CTC CAG (SEQ ID NO:_)            CTT GGT CCC-3′ aHJ_(K)-3: 5′-dTGG AATTCT CGA GAT TGC TAG TCA ACG TTT GAT ATC CAC (SEQ ID NO:_)            TTT GGT CCC-3′ aHJ_(K)-4: 5′-dTGG AATTCT CGA GAT TGC TAG TCA ACG TTT GAT CTC CAC (SEQ ID NO:_)            CTT GGT CCC-3′ aHJ_(K)-5: 5′-dTGG AATTCT CGA GAT TGC TAG TCA ACG TTT AAT CTC CAG (SEQ ID NO:_)            TCG TGT CCC-3′ Sense T7 & EcoRI-linked Primer: 5′-dCCA GTGAAT TGT AAT ACG ACT CAC TAT AGG GAA CGG (SEQ ID NO:_)     CAT GGAATG AAT TCC CCC CCC CC-3′ T7-Linked Antisense HJk Primers: aT7HJ_(K)-1:5′-dCCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GAA TGG (SEQ ID NO:_)             AAT TCG GCC CCC GAG GCC ACG TTT GAT TTC CAC CTT             GGT CCC-3′ aT7HJ_(K)-2:5′-dCCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GAA TGG (SEQ ID NO:_)             AAT TCG GCC CCC GAG GCC ACG TTT GAT CTC CAG CTT             GGT CCC-3′ aT7HJ_(K)-3:5′-dCCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GAA TGG (SEQ ID NO:_)             AAT TCG GCC CCC GAG GCC ACG TTT GAT ATC CAC TTT             GGT CCC-3′ aT7HJ_(K)-4:5′-dCCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GAA TGG (SEQ ID NO:_)             AAT TCG GCC CCC GAG GCC ACG TTT GAT CTC CAC CTT             GGT CCC-3′ aT7HJ_(K)-5:5′-dCCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GAA TGG (SEQ ID NO:_)             AAT TCG GCC CCC GAG GCC ACG TTT AAT CTC CAG TCG             TGT CCC-3′ Sense Peptide Linker-Linked HV_(K)-SpecificPrimers: sPLHV_(K)-1: 5′-TCC TCA GGC GGC GGC GGC TCT GGC GGA GGT GGC AGC(SEQ ID NO:_)              GGC GGT GGC GGA TCC GAC ATC CAG ATG ACC CAGTCT              CC-3′ sPLHV_(K)-2: 5′-TCC TCA GGC GGC GGC GGC TCT GGCGGA GGT GGC AGC (SEQ ID NO:_)              GGC GGT GGC GGA TCC GAT GTTGTG ATG ACT CAG TCT              CC-3′ sPLHV_(K)-3: 5′-TCC TCA GGC GGCGGC GGC TCT GGC GGA GGT GGC AGC (SEQ ID NO:_)              GGC GGT GGCGGA TCC GAA ATT GTG TTG ACG CAG TCT              CC-3′ sPLHV_(K)-4:5′-TCC TCA GGC GGC GGC GGC TCT GGC GGA GGT GGC AGC (SEQ ID NO:_)             GGC GGT GGC GGA TCC GAC ATC GTG ATG ACC CAG TCT             CC-3′ sPLHV_(K)-5: 5′-TCC TCA GGC GGC GGC GGC TCT GGC GGAGGT GGC AGC (SEQ ID NO:_)              GGC GGT GGC GGA TCC GAA ACG ACACTC ACG CAG TCT              CC-3′ sPLHV_(K)-6: 5′-TCC TCA GGC GGC GGCGGC TCT GGC GGA GGT GGC AGC (SEQ ID NO:_)              GGC GGT GGC GGATCC GAA ATT GTG CTG ACT CAG TCT              CC-3′

[0159] TABLE 3 Human V_(λ)-Specific Primers Antisense XhoI & StopCodon-Linked HJ_(λ)Primers: aHJ_(λ)-1: 5′-dTGG AAT TCT CGA GAT TGC TAGTCA ACC TAG GAC GGT (SEQ ID NO:_) GAC CTT GGT CCC-3′ aHJ_(λ)-2: 5′-dTGGAAT TCT CGA GAT TGC TAG TCA ACC TAG GAC GGT (SEQ ID NO:_) CAG CTT GGTCCC-3′ aHJ_(λ)-3: 5′-dTGG AAT TCT CGA GAT TGC TAG TCA ACC TAA AAC GGT(SEQ ID NO:_) GAG CTG GGT CCC-3′ Sense T7 & EcoRI-linked Primer: 5′-dCCAGTG AAT TGT AAT ACG ACT CAC TAT AGG GAA CGG (SEQ ID NO:_). CAT GGAATG AAT TCC CCC CCC CC-3′ T7-Linked Antisense HJ_(λ)Primers:aT7HJ_(λ)-1: 5′-dCCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GAA TGG (SEQ IDNO:_) AAT TCG GCC CCC GAG GCC ACC TAG GAC GGT GAC CTT GGT CCC-3′aT7HJ_(λ)-2: 5′-dCCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GAA TGG (SEQ IDNO:_) AAT TCG GCC CCC GAG GCC ACC TAG GAC GGT CAG CTT GGT CCC-3′aT7HJ_(λ)-3: 5′-dCCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GAA TGG (SEQ IDNO:_) AAT TCG GCC CCC GAG GCC ACC TAA AAC GGT GAG CTG GGT CCC-3′ SensePeptide Linker-Linked HV_(λ)Specific Primers: sPLHV_(λ)-1: 5′-TCC TCAGGC GGC GGC GGC TCT GGC GGA GGT GGC AGC (SEQ ID NO:_) GGC GGT GGC GGATCC CAG TCT GTG TTG ACG CAG CCG CC-3′ sPLHV_(λ)-2: 5′-TCC TCA GGC GGCGGC GGC TCT GGC GGA GGT GGC AGC (SEQ ID NO:_) GGC GGT GGC GGA TCC CAGTCT GCC CTG ACT CAG CCT GC-3′ sPLHV_(λ)-3: 5′-TCC TCA GGC GGC GGC GGCTCT GGC GGA GGT GGC AGC (SEQ ID NO:_) GGC GGT GGC GGA TCC TCC TAT GTGCTG ACT CAG CCA CC-3′ sPLHV_(λ)-4: 5′-TCC TCA GGC GGC GGC GGC TCT GGCGGA GGT GGC AGC (SEQ ID NO:_) GGC GGT GGC GGA TCC TCT TCT GAG CTG ACTCAG GAC CC-3′ sPLHV_(λ)-5: 5′-TCC TCA GGC GGC GGC GGC TCT GGC GGA GGTGGC AGC (SEQ ID NO:_) GGC GGT GGC GGA TCC CAC GTT ATA CTG ACT CAA CCGCC-3′ sPLHV_(λ)-6: 5′-TCC TCA GGC GGC GGC GGC TCT GGC GGA GGT GGC AGC(SEQ ID NO:_) GGC GGT GGC GGA TCC CAG GCT GTG CTC ACT CAG CCG TC-3′sPLHV_(λ)-7: 5′-TCC TCA GGC GGC GGC GGC TCT GGC GGA GGT GGC AGC (SEQ IDNO:_) GGC GGT GGC GGA TCC AAT TTT ATG CTG ACT CAG CCC CA-3′

[0160] Table 4 lists oligonucleotides that can be used for fusingvariable DNA homologs to form a single chain antibody as discussed inExample 10. TABLE 4 Human scF_(v) Primers Sense Sfi scF_(v) Primer:5′-TTG TTA TTA CTC GCG GCC CAG CCG GCC ATG GCA CAG GT-3′) (SEQ ID NO:_)Antisense Sfi scF_(v) Primer: 5′-GTC CTC GTC GAC TGG AAT TCG GCC CCC GAGGCC AC-3′) (SEQ ID NO:_)

[0161] Additional antisense primers can be designed and synthesized tohybridize to the constant region of either kappa or lambda mRNA toproduce the V_(K) or V_(λ) DNA homologs coding for constant region aminoacids of either kappa or lambda chain to produce an F_(ab) fragmentrather than an F_(V).

[0162] All primers and oligonucleotides used herein and shown on Tables1-3 are obtainable commercial customer oligonucleotide synthesiscompanies such as Invitrogen (Carlsbad, Calif.) or are synthesized on anApplied Biosystems DNA synthesizer, model 381A, using the instructionsand recommendations of the manufacturer.

Example 2

[0163] Preparation of Source mRNAs Containing the V_(H) and V_(L) GeneRepertoire

[0164] Total cellular RNA was prepared from the blood cells collectedfrom a pool of patients using the RNA preparation methods well known inthe art as described by Chomczynski et al., Anal Biochem., 162:156-159(1987) and the RNA isolation kit produced by QIAGEN GmbH (Hilden,Germany).

[0165] Messenger RNA (mRNA) enriched for sequences containing long polyA tracts was prepared from the total cellular RNA using methodsdescribed in “Molecular Cloning: A Laboratory Manual”, Maniatis et al.,eds., Cold Spring Harbor Laboratory, New York, (1982). Briefly, thetotal RNA isolated from the blood cells prepared as described above wasresuspended in 1 ml of DEPC-H₂O and maintained at 65° C. for 5 minutes.One ml of 2× high salt loading buffer consisting of 100 mM Tris-HCl, 1 Msodium chloride, 2.0 mM disodium ethylenediamine tetraacetic acid (EDTA)at pH 7.5, and 0.2% sodium dodecyl sulfate (SDS) was added to theresuspended RNA and the mixture allowed to cool to room temperature. Themixture was then applied to an oligo-dT (Collaborative Research Type 2or Type 3) column that was previously prepared by washing the oligo-dTwith a solution containing 0.1 M sodium hydroxide and 5 mM EDTA and thenequilibrating the column with DEPC-H₂O. The eluate was collected in asterile polypropylene tube and reapplied to the same column afterheating the eluate for 5 minutes at 65° C. The oligo-dT column was thenwashed with 2 ml of high salt loading buffer consisting of 50 mMTris-HCl at pH 7.5, 500 mM sodium chloride, 1 mM EDTA at pH 7.5 and 0.1%SDS. The oligo-dT column was then washed with 2 ml of 1× medium saltbuffer consisting of 50 mM Tris-HCl at pH 7.5, 100 mM sodium chloride, 1mM EDTA and 0.1% SDS. The messenger RNA was eluted from the oligo-dTcolumn with 1 ml of buffer consisting of 10 mM Tris-HCl at pH 7.5, 1 mMEDTA at pH 7.5 and 0.05% SDS. The messenger RNA was purified byextracting this solution with phenol/chloroform followed by a singleextraction with 100% chloroform. The messenger RNA was concentrated byethanol precipitation and resuspended in DEPC H₂O.

[0166] The messenger RNA isolated by the above process contains aplurality of different V_(H) and V_(L) mRNA transcripts, i.e., greaterthan about 10⁴ different V_(H) and V_(L) gene repertoires.

Example 3

[0167] Transcriptional Amplification of the V_(H) Gene Repertoire

[0168] Transcriptional amplification is performed using the scheme asdepicted in FIG. 4. In detail, about 5-10 μg of poly (A)⁺ mRNAs inDEPC-treated water were first hybridized (annealed) with 1 μM antisenseprimer mixture comprising equal amounts of antisense EcoRI & stopcodon-linked HJ_(H) primers, for example, aHJ_(H)-1, 5′-dTGG AAT GAA TTCGAT TGC TAG TCA GAC GGT GAC CAG GGT GCC-3′ (SEQ ID NO:__; aHJ_(H)-2,5′-dTGG AAT GAA TTC GAT TGC TAG TCA GAC GGT GAC CAT TGT CCC-3′ (SEQ IDNO:__; aHJ_(H)-3, 5′-dTGG AAT GAA TTC GAT TGC TAG TCA GAC GGT GAC CAGGGT TCC-3′ (SEQ ID NO:__; and aHJ_(H)-4, 5′-dTGG AAT GAA TTC GAT TGC TAGTCA GAC GGT GAC CGT GGT CCC-3′ (SEQ ID NO:__) as listed in Table 1 (theEcoRI site and stop codons in three different frames are underlined), at65° C. for 5 minutes and then cooled down to room temperature. Themixture was subsequently added to a reverse transcription (RT) reactionadmixture (20 μl) on ice, comprising 2 μl of 10× buffer (400 mMTris-HCl, pH 8.3 at 25° C., 300 mM KCl, 80 mM MgCl₂, 2 M betaine, 100 mMDTT), dNTPs (1.5 mM each for dATP, dGTP, dCTP and dTTP) and RNaseinhibitors (20 U). After M-MuLV reverse transcriptase (40 U) was added,the reaction was incubated at 42° C. for 1 hour and shifted to 52° C.for another 15 min. The first-strand cDNAs so obtained were collected bya microcon-50 microconcentrater filter and then mixed with terminaltransferase (50 U), dGTP (1.5 mM) in 0.5× buffer. The reaction wasincubated at 37° C. for 15 min, stopped by denaturation at 94° C. for 3min and instantly mixed with 1 μM sense T7 & NotI-linked primer of5′-dCCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GAA CGG CAT GGA ATG CGG CCGCCC CCC CCC C-3′ (SEQ ID NO:__), the NotI site is underlined. Afterbriefly centrifuging, Taq DNA polymerase (3.5 U) and dNTPs (1.5 mM eachfor dATP, dCTP and dTTP) were added to form T7 promoter-linkeddouble-stranded cDNAs at 52° C. for 3 min and then 68° C. for 10 min.The resulting ds cDNAs comprise ds V_(H) DNA homologs ready as templatefor RNA transcription amplification and in vitro transcription andtranslation. An in vitro transcription (IVT) reaction (40 μl) wasprepared, containing 4 μl of 10× buffer, above reaction, rNTPs (2 mMeach for ATP, GTP, CTP and UTP), and T7 RNA polymerase (160 U). After 1hour incubation at 37° C., the amplified sense RNA transcripts were useddirectly for another round of amplification by repeating the aboveprocedure, however, without the tailing reaction. The quality ofamplified RNA library (2 μg) was assessed on a 1% formaldehyde-agarosegel. The resulting RNA transcripts can be translated into polypeptidesof V_(H) polypeptides.

[0169] The double stranded V_(H) DNA homologs made by above protocolcontain at the 5′ end of the NotI restriction enzyme site and at the 3′end the EcoRI restriction site for cloning into a vector predigestedwith the NotI & EcoRI, in this instance, the Lambda ZAP II vector fromStratagene (La Jolla, Calif.) was predigested with NotI and EcoRI andthe V_(H) DNA homologs so made were digested with NotI and EcoRI andligated into the Lambda ZAP II vector at the NotI and EcoRI sites. TheV_(H) DNA homologs so prepared also encode stop codons (UAA, UAG andUGA) in three different frames at the 3′ end as in vitro and/or in vivotranslation stop codons. The V_(H) DNA homologs contain the 5′ terminalsequences, such as the ribosome binding site, translation initiationsite and ATG start codon, derived from the original heavy chain mRNAtranscripts, and thus, can be ranslated into the V_(H) polypeptides invivo or in vitro directly.

[0170] The V_(H) DNA homologs so made contain the T7 promoter sequenceat the 5′ end and can be further transcriptionally amplified byrepeating the IVT procedure using the T7 RNA polymerase. The resultingds V_(H) DNA homologs can be the templates for further cloning processinto either in vivo expression vector or in vitrotranscription/translation unit sequences in the PROfusion or ribosomedisplay methods.

Example 4

[0171] Transcriptional Amplification of the V_(K) Gene Repertoire

[0172] In this example, the transcriptional amplification is performedusing the scheme as depicted in FIG. 4. In detail, 5-10 μg of poly (A)⁺mRNAs in DEPC-treated water were first hybridized (annealed) with 1 tMantisense primer mixture comprising equal amounts of antisense XhoI &stop codon-linked HJ_(k) primers, for example, aHJ_(k)-1, 5′-dTGG AATTCT CGA GAT TGC TAG TCA ACG TTT GAT TTC CAC CTT GGT CCC-3′ (SEQ IDNO:__); aHJ_(K)-2, 5′-dTGG AAT TCT CGA GAT TGC TAG TCA ACT TTT GAT CTCCAG CTT GGT CCC-3′ (SEQ ID NO:__); aHJ_(K)-3, 5′-dTGG AAT TCT CGA GATTGC TAG TCA ACG TTT GAT ATC CAC TTT GGT CCC-3′ (SEQ ID NO:__);aHJ_(K)-4, 5′-dTGG AAT TCT CGA GAT TGC TAG TCA ACG TTT GAT CTC CAC CTTGGT CCC-3′ (SEQ ID NO:__); and aHJ_(K)-5, 5′-dTGG AAT TCT CGA GAT TGCTAG TCA ACG TTT AAT CTC CAG TCG TGT CCC-3′ (SEQ ID NO:__) as listed inTable 2 (the XhoI site and stop codons in three different frames areunderlined), at 65° C. for 5 minutes and then cooled down to roomtemperature. The mixture was subsequently added to a reversetranscription (RT) reaction admixture (20 μl) on ice, comprising 2 μl of10× buffer (400 mM Tris-HCl, pH 8.3 at 25° C., 300 mM KCl, 80 mM MgCl₂,2 M betaine, 100 mM DTT), dNTPs (1.5 mM each for dATP, dGTP, dCTP anddTTP) and RNase inhibitors (20 U). After M-MuLV reverse transcriptase(40 U) was added, the reaction was incubated at 42° C. for 1 hour andshifted to 52° C. for another 15 min. The first-strand cDNAs so obtainedwere collected by a microcon-50 microconcentrater filter and then mixedwith terminal transferase (50 U), dGTP (1.5 mM) in 0.5× buffer. Thereaction was incubated at 37° C. for 15 min, stopped by denaturation at94° C. for 3 min and instantly mixed with 1 μM sense T7 & EcoRI-linkedprimer, for example, 5′-dCCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GAA CGGCAT GGA ATG AAT TCC CCC CCC CC-3′ (SEQ ID NO:__). After brieflycentrifuiging, Taq DNA polymerase (3.5 U) and dNTPs (1.5 mM each fordATP, dCTP and dTTP) were added and the mixture incubated at 52° C. for3 min and then 68° C. for 10 min to form T7 promoter-linkeddouble-stranded cDNAs. The resulting ds cDNAs comprise ds V_(K) DNAhomologs ready as template for RNA transcription amplification and invitro transcription and translation. An in vitro transcription (IVT)reaction (40 μl) was prepared, containing 4 μl of 10× buffer, abovereaction, rNTPs (2 mM each for ATP, GTP, CTP and UTP), and T7 RNApolymerase (160 U). After 1 hour incubation at 37° C., the amplifiedsense RNA transcripts were used directly for another round ofamplification by repeating the above procedure, however, without thetailing reaction. The quality of amplified RNA library (2 μg) wasassessed on a 1% formaldehyde-agarose gel. The resulting RNA transcriptscan be translated into polypeptides of V_(K) polypeptides.

[0173] The double stranded V_(K) DNA homologs made by above protocolcontain at the 5′ end an EcoRI restriction enzyme site and at the 3′ endan XhoI restriction site for cloning into a vector predigested with theEcoRI and XhoI, for example, the Lambda ZAP II vector from Stratagene(La Jolla, Calif.) was predigested with EcoRI and XhoI, and the V_(K)DNA homologs so made were digested with EcoRI and XhoI and ligated intothe Lambda ZAP II vector at the EcoRI and XhoI sites. The V_(K) DNAhomologs so prepared also encode stop codons (UAA, UAG and UGA) in threedifferent frames at the 3′ end as in vitro and/or in vivo translationstop codons. The V_(K) DNA homologs contain 5′ terminal sequences suchas the ribosome binding site, transtation initiation site and ATG startcodon derived from the original light kappa chain mRNA transcripts, andthus, can be translated into the V_(K) polypeptides in vivo or in vitrodirectly.

[0174] The V_(K) DNA homologs so made contain the T7 promoter sequenceat the 5′ end and can be further transcriptionally amplified byrepeating the IVT using the T7 RNA polymerase. The resulting ds V_(K)DNA homologs can be the templates for further cloning into either an invivo expression vector or in an in vitro transcription/translationsystem such as in the PROfusion or ribosome display methods.

Example 5

[0175] Transcriptional Amplification of the V_(λ) Gene Repertoire

[0176] In this example, the transcriptional amplification is performedusing the scheme as depicted in FIG. 4. In detail, about 5-10 μg of poly(A)⁺ mRNAs in DEPC-treated water were first hybridized (annealed) with 1μM antisense primer mixture comprising equal amounts of antisense XhoI &stop codon-linked HJ_(λ) primers, for example, aHJ_(λ)-1, 5′-dTGG AATTCT CGA GAT TGC TAG TCA ACC TAG GAC GGT GAC CTT GGT CCC-3′ (SEQ IDNO:__); HJ_(λ)-2, 5′-dTGG AAT TCT CGA GAT TGC TAG TCA ACC TAG GAC GGTCAG CTT GGT CCC-3′ (SEQ ID NO:__); and HJ_(λ)-3, 5′-dTGG AAT TCT CGA GATTGC TAG TCA ACC TAA AAC GGT GAG CTG GGT CCC-3′ (SEQ ID NO:__) as listedin Table 3 (the XhoI site and the stop codons in three different framesare underlined), at 65° C. for 5 minutes and then cooled down to roomtemperature. The mixture was subsequently added to a reversetranscription (RT) reaction admixture (20 μl) on ice, comprising 2 μl of10× buffer (400 mM Tris-HCl, pH 8.3 at 25° C., 300 mM KCl, 80 mM MgCl₂,2 M betaine, 100 mM DTT), dNTPs (1.5 mM each for dATP, dGTP, dCTP anddTTP) and RNase inhibitors (20 U). After M-MuLV reverse transcriptase(40 U) was added, the reaction was incubated at 42° C. for 1 hour andshifted to 52° C. for another 15 min. The first strand cDNAs so obtainedwere collected by a microcon-50 microconcentrater filter and then mixedwith terminal transferase (50 U), dGTP (1.5 mM) in 0.5× buffer. Thereaction was incubated at 37° C. for 15 min, stopped by denaturation at94° C. for 3 min and instantly mixed with 1 μM sense T7 & EcoRI-linkedprimer, such as 5′-dCCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GAA CGG CATGGA ATG AAT TCC CCC CCC CC-3′ (SEQ ID NO:__). After brieflycentrifuging, Taq DNA polymerase (3.5 U) and dNTPs (1.5 mM each fordATP, dCTP and dTTP) were added and the mixture incubated at 52° C. for3 min and then 68° C. for 10 min to form T7 promoter—linkeddouble-stranded cDNAs. The resulting ds cDNAs comprise ds V_(λ) DNAhomologs ready as template for RNA transcription amplification and invitro transcription and translation. An in vitro transcription (IVT)reaction (40 μl) was prepared, containing 4 μl of 10× buffer, abovereaction, rNTPs (2 mM each for ATP, GTP, CTP and UTP), and T7 RNApolymerase (160 U). After one hour incubation at 37° C., the amplifiedsense RNA transcripts were used directly for another round ofamplification by repeating the above procedure, however, without thetailing reaction. The quality of amplified RNA library (2 μg) wasassessed on a 1% formaldehyde-agarose gel. The resulting RNA transcriptscan be translated into polypeptides of V_(λ) polypeptides.

[0177] The double stranded V_(λ) DNA homologs made by above protocolcontain at the 5′ end an EcoRI restriction enzyme site and at the 3′end, an XhoI restriction site for cloning into a vector predigested withthe EcoRI and XhoI. For example, the Lambda ZAP II vector fromStratagene (La Jolla, Calif.) was predigested with EcoRI and XhoI andthe V_(λ) DNA homologs so made were digested with EcoRI and XhoI, andligated into the Lambda ZAP II vector at the EcoRI and XhoI sites. TheV_(λ) DNA homologs so prepared also encode stop codons (UAA, UAG andUGA) in three different frames at the 3′ end and are in vitro and/or invivo translation stop codons. The V_(λ) DNA homologs contains 5′sequences such as ribosome binding site, translation initiation site andATG start codon derived from the original light lambda chain mRNAtranscripts. Thus, the instant clones can be translated into the V_(λ)polypeptides in vivo or in vitro directly.

[0178] The V_(λ) DNA homologs so made contain the T7 promoter sequenceat the 5′ end and can be further transcriptionally amplified byrepeating the IVT using the T7 RNA polymerase. The resultant ds V_(λ)DNA homologs can be the templates for further cloning into either invivo expression vector or in vitro transcription/translation unitsequences in the PROfusion or ribosome display methods.

Example 6

[0179] Human V_(H) Expression Phage Library Construction

[0180] The Lambda ZAP II™ vector from Stratagene (La Jolla, Calif.)(Short et al., Nucleic Acids Res., 16:7583-7600, 1988) was used as anexample of an expression vector system for constructing theV_(H)-expressing library. The Lambda ZAP II™ vector is well known to theskilled in the art as a phage vector that can be efficiently packaged invitro and reintroduced into bacterial host cells. The expressed proteintherefrom can be detected at the level of single phage plaques. Thesignal to noise ratio for screening of phage libraries is very high withvery low nonspecific binding. Finally, the vector can be converted by invivo excision into a phagemid vector for further analysis such as suchas sequencing analysis of isolated clones.

[0181] To prepare an expression library of the V_(H) gene repertoire asdepicted in FIG. 7, the V_(H) DNA homologs enriched in V_(H) generepertoire sequences were prepared according to Example 3. These doublestranded V_(H) DNA homologs, containing NotI and EcoRI restrictionenzyme sites, were digested with the restriction enzymes NotI and EcoRI.The digested V_(H) DNA homologs were subject to electrophoresis andpurified on a 1% agarose gel using the standard electroelution techniquedescribed in “Molecular Cloning: A Laboratory Manual”, Maniatis et al.,eds., Cold Spring Harbor, N.Y., (1982). The region of the gel containingDNA fragments of approximately 350 bps was excised, electroeluted into adialysis membrane, ethanol precipitated and resuspended in 10 mMTris-HCl pH 7.5 and 1 mM EDTA to a final concentration of 10 ng/ul.Equimolar amounts of the V_(H) DNA homologs insert were then ligatedovernight at 5° C. to 1 μg of Lambda ZAP II vector previously cut byEcoRI and NotI. The ligation mixture containing the V_(H) DNA homologswere packaged according to the manufacturer specifications usingGigapack Gold II Packing Extract (Stratagene Cloning Systems, La Jolla,Calif.). The expression libraries were then ready to be plated on XL-1Blue cells.

Example 7

[0182] Human V_(K) Expression Phage Library Construction

[0183] To prepare an expression library of the V_(K) gene repertoire asdepicted in FIG. 8, the V_(K) DNA homologs enriched in V_(K) generepertoire sequences were prepared according to Example 4. These doublestranded V_(K) DNA homologs, containing EcoRI and XhoI restrictionenzyme sites, were digested with the restriction enzymes EcoRI and XhoI.The digested V_(K) DNA homologs were subjected to electrophoresis andpurified on a 1% agarose gel using the standard electroelution techniquedescribed in “Molecular Cloning: A Laboratory Manual”, Maniatis et al.,eds., Cold Spring Harbor, N.Y., (1982). The region of the gel containingDNA fragments of approximately 350 bps was excised, electroeluted into adialysis membrane, ethanol precipitated and resuspended in 10 mMTris-HCl pH 7.5 and 1 mM EDTA to a final concentration of 10 ng/ul.Equimolar amounts of the V_(K) DNA homologs insert were then ligatedovernight at 5° C. to 1 μg of Lambda ZAP II vector previously cut byEcoRI and XhoI. The ligation mixture containing the V_(K) DNA homologswere packaged according to the manufacturer specifications usingGigapack Gold II Packing Extract (Stratagene Cloning Systems, La Jolla,Calif.). The expression libraries were then ready to be plated on XL-1Blue cells.

Example 8

[0184] Human V_(λ) Expression Phage Library Construction

[0185] To prepare an expression library of the V_(λ) gene repertoire,the V_(λ) DNA homologs enriched in V_(λ) gene repertoire sequences wereprepared according to Example 5. These double stranded V_(λ) DNAhomologs, containing EcoRI and XhoI restriction enzyme sites, weredigested with the restriction enzymes EcoRI and XhoI. The digested V_(λ)DNA homologs were subject to electrophoresis and purified on a 1%agarose gel using the standard electroelution technique described in“Molecular Cloning: A Laboratory Manual”, Maniatis et al., eds., ColdSpring Harbor, N.Y., (1982). The region of the gel containing DNAfragments of approximately 350 bps was excised, electroeluted into adialysis membrane, ethanol precipitated and resuspended in 10 mMTris-HCl pH 7.5 and 1 mM EDTA to a final concentration of 10 ng/ul.Equimolar amounts of the V_(λ) DNA homologs insert were then ligatedovernight at 5° C. to 1 ug of Lambda ZAP II vector previously cut byEcoRI and XhoI. The ligation mixture containing the VA DNA homologs werepackaged according to the manufacturers specifications using GigapackGold II Packing Extract (Stratagene Cloning Systems, La Jolla, Calif.).The expression libraries were then ready to be plated on XL-1 Bluecells.

Example 9

[0186] Human V_(H)+V_(L) Combinatorial Antibody Expression Phage LibraryConstruction

[0187] The construction of a combinatorial library of human antibodieswas accomplished by combining the V_(H) library made in Example 6 witheither one or both of the V_(K) library made in Example 7 and the V_(λ)library made in Example 8 at the symmetric EcoRI sites present in eachvector as depicted in FIG. 9. This resulted in a library of clones, eachof which potentially co-expresses a V_(H) and a V_(L) gene on a singletranscript chain. And each host cell may express a heterodimericantibody consisting of a V_(H) polypeptide and V_(L) polypeptide.

[0188] The phage library DNA of V_(H) and V_(L) (V_(κ) and V_(λ)) wasfirst purified from each library. The phage libraries prepared inExample 6, 7 and 8 were amplified and 500 μg of phage library DNAprepared from the amplified phage stock using the procedures describedin “Molecular Cloning: A Laboratory Manual”, Maniatis et al., eds., ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982).

[0189] To accomplish the cross at the EcoRI site as depicted in FIG. 9,the V_(H) phage library DNA as made according to Example 6 was digestedwith HindIII, the resulting 5′ ends dephosphorylated and the productfurther digested with EcoRI. This process cleaved the right arm of theLambda ZAPII vector into several pieces but the left arm containing theV_(H) sequences, remained intact. Fifty (50) μg of V_(H)-expressionlibrary phage DNA were maintained in a solution containing 100 units ofHindIII (Boehringer Mannheim, Indianapolis, Ind.) in 200 μl of a buffersupplied by the endonuclease manufacturer for 1.5 hours at 37° C. Thesolution was then extracted with a mixture of phenol and chloroform. TheDNA was then ethanol precipitated and resuspended in 100 μl of water.This solution was admixed with 100 units of the restriction endonucleaseEcoRI (Boehringer Mannheim, Indianapolis, Ind.) in a final volume of 200μl of buffer containing the components specified by the manufacturer.This solution was maintained at 37° C. for 1.5 hours and the solutionwas then extracted with a mixture of phenol and chloroform. The DNA wasethanol precipitated and the DNA resuspended in TE.

[0190] In a parallel fashion, the phage library DNA of V_(L) (V_(K) andV_(λ)) was digested with MluI, dephosphorylated and further digestedwith EcoRI, destroying the left arm of the Lambda ZAPII but leaving theright arm containing the V_(L) sequences intact. The V_(L) expressionlibrary prepared in Example 7 (the V_(K)) and Example 8 (the V_(λ)) wereamplified. Twenty five (25) μg of each of the V_(K) and V_(λ) expressionlibrary phage DNAs were mixed and maintained in a solution containing100 units of MluI restriction endonuclease (Boehringer Mannheim,Indianapolis, Ind.) in 200 μl of a buffer supplied by the endonucleasemanufacturer for 1.5 hours at 37° C. The solution was then extractedwith a mixture of phenol and chloroform saturated with 0.1 M Tris-HCl atpH 7.5. The DNA was then ethanol precipitated and resuspended in 100 μlof water. This solution was admixed with 100 units of EcoRI (BoehringerMannheim, Indianapolis, Ind.) in a final volume of 200 μl of buffercontaining the components specified by the manufacturer. This solutionwas maintained at 37° C. for 1.5 hours and the solution was thenextracted with a mixture of phenol and chloroform. The DNA was ethanolprecipitated and the DNA resuspended in TE.

[0191] The restriction digested V_(H) and V_(L) expression librarieswere ligated together. The ligation reaction consisted of 1 μg of V_(H)and 1 μg of V_(L) phage library DNA in a 10 μl reaction using thereagents supplied in a ligation kit purchased from Stratagene CloningSystems (La Jolla, Calif.). After ligation, only clones which resultedfrom combination of a left arm of V_(H)-containing clones and a rightarm of V_(L)-containing clones reconstituted a viable phage. Theligation mixture containing the V_(H)+V_(L) DNA homologs were packagedaccording to the specifications of the manufacturer using the GigapackGold II Packing Extract (Stratagene Cloning Systems, La Jolla, Calif.).The V_(H)+V_(L)-expressing libraries were then ready to be plated onXL-1 Blue cells.

Example 10

[0192] Single Chain Human Antibody Library Construction

[0193] The single chain antibody or scF_(v) typically has a linkerpeptide such as (GlyGlyGlyGlySer)_(n) linking the V_(H) and V_(L) genes.The construction of an scF_(v) human antibody library can be made bylinking the V_(H) and V_(L) DNA homologs together with a sequenceencoding a linking peptide and inserting the single chain(V_(H)+linker+V_(L)) into an appropriate expression vector or in vitrotranscription/translation unit sequence as depicted in FIG. 10.

[0194] The amplification strategy for the V_(H) and V_(L) (V_(K) andV_(λ)) DNA homologs is depicted in FIG. 2, wherein the sequence-specificantisense primers are linked with a T7 RNA promoter sequence and thesense primers are sequence-specific primers. The antisense primers forthe V_(H) DNA homologs and the sense sequence-specific primers for theV_(L) have incorporated the linker sequence so that the V_(H) and V_(L)DNA homologs can be overlapped and operatively linked by the linkersequence into a single chain sequence of V_(H)+V_(L) DNA homologs. Thesingle chain V_(H)+V_(L) DNA homologs can be amplified with two flankingprimers with appropriate restriction enzyme sites for inserting into anappropriate expression vector or in vitro transcription/translation unitfor expressing the scF_(v) antibody.

[0195] The transcriptional amplification of the V_(H) gene is performedusing the scheme as depicted in FIG. 2. In detail, about 5-10 μg of poly(A)⁺ mPNAs in DEPC-treated water were first hybridized (annealed) with 1μM antisense primer mixture comprising equal amounts of T7 & peptidelinker (PL)-linked antisense T7 HJ_(H) primers, such as aT7PLHJ_(H)-1(5′-dCCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GAA AGA GCC GCC GCC GCC TGAGGA GAC GGT GAC CAG GGT GCC-3′) (SEQ ID NO:__); aT7PLHJ_(H)-2 (5′-dCCAGTG AAT TGT AAT ACG ACT CAC TAT AGG GAA AGA GCC GCC GCC GCC TGA GGA GACGGT GAC CAT TGT CCC-3′) (SEQ ID NO:__); aT7PLHJ_(H)-3 (5′-dCCA GTG AATTGT AAT ACG ACT CAC TAT AGG GAA AGA GCC GCC GCC GCC TGA GGA GAC GGT GACCAG GGT TCC-3′) (SEQ ID NO:__); and aT7PLHJ_(H)-4 (5′-dCCA GTG AAT TGTAAT ACG ACT CAC TAT AGG GAA AGA GCC GCC CC GCC TGA GGA GAC GGT GAC CGTGGT CCC-3′) (SEQ ID NO:__) as in Table 1 (the overlapping peptide linkeris underlined), at 65° C. for 5 minutes and cooled to room temperature.The mixture was subsequently added to a reverse transcription (RT)reaction admixture (20 μl) on ice, comprising 2 μl of 10× buffer (400 mMTris-HCl, pH 8.3 at 25° C., 300 mM KCl, 80 mM MgCl₂, 2 M betaine, 100 mMDTT), dNTPs (1.5 mM each for dATP, dGTP, dCTP and dTTP) and RNaseinhibitors (20 U). After M-MuLV reverse transcriptase (40 U) was added,the reaction was incubated at 42° C. for 1 hour and shifted to 52° C.for another 15 min. The first-strand cDNAs so obtained were collected bya microcon-50 microconcentrater filter. The resultant purified firststrand cDNAs were denatured from the mRNAs by denaturation at 94° C. for3 min and instantly mixed with 1 μM of a sense HV_(H) primer mixturecomprising equal amounts of sense HV_(H) primers such as sHV_(H)-1(5′-CAG CCG GCC ATG GCA CAG GTN CAG CTG GTR CAG TCT GG-3′) (SEQ IDNO:__); sHV_(H)-2 (5′-CAG CCG GCC ATG GCA CAG GTC CAG CTG GTR CAG TCTGGG G-3′) (SEQ ID NO:__); sHV_(H)-3 (5′-CAG CCG GCC ATG GCA CAG GTK CAGCTG GTG SAG TCT GGG-3′) (SEQ ID NO:__); sHV_(H)-4 (5′-CAG CCG GCC ATGGCA CAG GTC ACC TTG ARG GAG TCT GGT CC-3′) (SEQ ID NO:__); sHV_(H)-5(5′-CAG CCG GCC ATG GCA CAG GTG CAG CTG GTG GAG WCT GG-3′) (SEQ IDNO:__); sHV_(H)-6 (5′-CAG CCG GCC ATG GCA CAG GTG CAG CTG GTG SAG TCYGG-3′) (SEQ ID NO:__); sHV_(H)-7 (5′-CAG CCG GCC ATG GCA CAG GTG CAG CTGCAG GAG TCG G-3′) (SEQ ID NO:__); sHV_(H)-8 (5′-CAG CCG GCC ATG GCA CAGGTG CAG CTG TTG SAG TCT G-3′) (SEQ ID NO:__); sHV_(H)-9 (5′-CAG CCG GCCATG GCA CAG GTG CAG CTG GTG CAA TCT G-3′), (SEQ ID NO:__); sHV_(H)-10(5′-CAG CCG GCC ATG GCA CAG GTG CAG CTG CAG GAG TCC GG-3′) (SEQ IDNO:__); sHV_(H)-11 (5′-CAG CCG GCC ATG GCA CAG GTG CAG CTA CAG CAG TGGG-3′) (SEQ ID NO:__); and sHV_(H)-12 (5′-CAG CCG GCC ATG GCA CAG GTA CAGCTG CAG CAG TCA G-3′) (SEQ ID NO:__).

[0196] After briefly centrifuging, Taq DNA polymerase (3.5 U) and dNTPs(1.5 mM each for dATP, dCTP, dGTP and dTTP) were added to formpromoter-linked double-stranded cDNAs at 52° C. for 3 min and then 68°C. for 10 min. An in vitro transcription (IVT) reaction (40 μl) wasprepared, containing 4 μl of 10× buffer, rNTPs (2 mM each for ATP, GTP,CTP and UTP), and T7 RNA polymerase (160 U). After 1 hour incubation at37° C., the amplified antisense V_(H) RNA transcripts were directly usedfor making the double stranded V_(H) DNA homologs. The antisense RNAtranscripts were purified and collected by a microcon-50microconcentrater filter and then subjected to a reverse transcription(RT) reaction admixture (20 μl), comprising 2 μl of 10× buffer, 1 μMsense HV_(H) primers as described in the above paragraph, dNTPs (1.5 mMeach for dATP, dGTP, dCTP and dTTP) and RNase inhibitors (20 U). AfterM-MuLV reverse transcriptase (40 U) was added, the reaction wasincubated at 42° C. for 1 hour and shifted to 52° C. for another 15 min.The sense orientation first-strand cDNAs so obtained were collected by amicrocon-50 microconcentrater filter. The resulting purified senseorientation first strand cDNAs were denatured from the antisense RNAtranscripts by denaturation at 94° C. for 3 min and instantly mixed with1 μM of a T7 & peptide linker-linked antisense HJ_(H) primer mixture asdescribed in the above paragraph. After briefly centrifuging, Taq DNApolymerase (3.5 U) and dNTPs (1.5 mM each for dATP, dCTP, dGTP and dTTP)were added to form promoter-linked double-stranded cDNAs at 52° C. for 3min and then 68° C. for 10 min.

[0197] The resulting ds V_(H) DNA homologs can be furthertranscriptionally amplified by repeating the IVT using the T7 RNApolymerase. The resultant ds V_(H) DNA homologs can be the templates forfurther cloning and processing into either an in vivo expression vectoror an in vitro transcription/translation system as in the PROfusion orribosome display method.

[0198] In the case of making the scF_(v) antibody, the antisenseamplified V_(H) RNA transcripts made by the steps above can be used asthe template for making single stranded sense V_(H) sequences by using asingle sense primer sequence, such as, the sense Sfi-scFv primer of5′-TTG TTA TTA CTC GCG GCC CAG CCG GCC ATG GCA CAG GT-3′) (SEQ ID NO.31) (Table 4) in a reverse transcription reaction. Briefly, theantisense RNA transcripts were purified and collected by a microcon-50microconcentrater filter and then subjected to the reverse transcription(RT) reaction admixture (20 μl), comprising 2 μl of 10× buffer, 1 μMsense Sfi-scFv primer as described in the above, dNTPs (1.5 mM each fordATP, dGTP, dCTP and dTTP) and RNase inhibitors (20 U). After M-MuLVreverse transcriptase (40 U) was added, the reaction was incubated at42° C. for 1 hour and shifted to 52° C. for another 15 min. Thesense-orientation first-strand cDNAs so obtained were collected by amicrocon-50 microconcentrater filter. The resulting purified senseorientation first strand cDNAs were denatured from the antisense RNAtranscripts by denaturation at 94° C. for 3 min. The single strandedantisense V_(H) DNA sequences were then used in an overlapping strandextension reaction with the single-stranded antisense V_(L) DNAsequences as described below.

[0199] The transcriptional amplification of a V_(K) gene is performedusing the scheme as depicted in FIG. 2. In detail, about 5-10 82 g ofpoly (A)⁺ mRNAs in DEPC-treated water were first hybridized (annealed)with 1 μM antisense primer mixture comprising equal amounts of T7-linkedantisense HJ_(k) primers such as aT7HJ_(K)-1 (5′-dCCA GTG AAT TGT AATACG ACT CAC TAT AGG GAA TGG AAT TCG GCC CCC GAG GCC ACG TTT GAT TTC CACCTT GGT CCC-3′) (SEQ ID NO:__); aT7HJ_(K)-2 (5′-dCCA GTG AAT TGT AAT ACGACT CAC TAT AGG GAA TGG AAT TCG GCC CCC GAG GCC ACG TTT GAT CTC CAG CTTGGT CCC-3′) (SEQ ID NO:__); aT7HJ_(K)-3 (5′-dCCA GTG AAT TGT AAT ACG ACTCAC TAT AGG GAA TGG AAT TCG GCC CCC GAG GCC ACG TTT GAT ATC CAC TTT GGTCCC-3′) (SEQ ID NO:__); aT7HJ_(K)-4 (5′-dCCA GTG AAT TGT AAT ACG ACT CACTAT AGG GAA TGG AAT TCG GCC CCC GAG GCC ACG TTT GAT CTC CAC CTT GGTCCC-3′) (SEQ ID NO:__); and aT7HJ_(K)-5 (5′-dCCA GTG AAT TGT AAT ACG ACTCAC TAT AGG GAA TGG AAT TCG GCC CCC GAG GCC ACG TTT AAT CTC CAG TCG TGTCCC-3′) (SEQ ID NO:__) (Table 2), at 65° C. for 5 minutes and cooleddown to room temperature. The reaction was subsequently were added to areverse transcription (RT) reaction admixture (20 μl) on ice, comprising2 μl of 10× buffer (400 mM Tris-HCl, pH 8.3 at 25° C., 300 mM KCl, 80 mMMgCl₂, 2 M betaine, 100 mM DTT), the above reaction mixture, dNTPs (1.5mM each for dATP, dGTP, dCTP and dTTP) and RNase inhibitors (20 U).After M-MuLV reverse transcriptase (40 U) was added, the reaction wasincubated at 42° C. for 1 hour and shifted to 52° C. for another 15 min.The first-strand cDNAs so obtained were collected by a microcon-50microconcentrater filter. The resulting purified first strand cDNAs weredenatured from the mRNAs by denaturation at 94° C. for 3 min andinstantly mixed with 1 μM of sense HVk primers such as sense peptidelinker-linked HVk primers such as sPLHV_(K)-1 (5′-TCC TCA GGC GGC GGCGGC TCT GGC GGA GGT GGC AGC GGC GGT GGC GGA TCC GAC ATC CAG ATG ACC CAGTCT CC-3′) (SEQ ID NO:__); sPLHV_(K)-2 (5′-TCC TCA GGC GGC GGC GGC TCTGGC GGA GGT GGC AGC GGC GGT GGC GGA TCC GAT GTT GTG ATG ACT CAG TCTCC-3′) (SEQ ID NO:__); sPLHV_(K)-3 (5′-TCC TCA GGC GGC GGC GGC TCT GGCGGA GGT GGC AGC GGC GGT GGC GGA TCC GAA ATT GTG TTG ACG CAG TCT CC-3′)(SEQ ID NO:__); sPLHV_(K)-4 (5′-TCC TCA GGC GGC GGC GGC TCT GGC GGA GGTGGC AGC GGC GGT GGC GGA TCC GAC ATC GTG ATG ACC CAG TCT CC-3′) (SEQ IDNO:__); sPLHV_(K)-5 (5′-TCC TCA GGC GGC GGC GGC TCT GGC GGA GGT GGC AGCGGC GGT GGC GGA TCC GAA ACG ACA CTC ACG CAG TCT CC-3′) (SEQ ID NO:__);and sPLHV_(K)-6 (5′-GGC AGC GGC GGT GGC GGA TCC GAA ATT GTG CTG ACT CAGTCT CC-3′) (SEQ ID NO:__) (Table 2). After briefly centrifuging, Taq DNApolymerase (3.5 U) and dNTPs (1.5 mM each for dATP, dCTP, dGTP and dTTP)were added to form promoter-linked double-stranded cDNAs at 52° C. for 3min and then 68° C. for 10 min. An in vitro transcription (IVT) reaction(40 μl) was prepared, containing 4 μl of 10× buffer, above reaction,rNTPs (2 mM each for ATP, GTP, CTP and UTP), and T7 RNA polymerase (160U). After one hour incubation at 37° C., the antisense amplified V_(K)RNA transcripts were directly used for making the double stranded V_(K)DNA homologs. The antisense RNA transcripts were purified and collectedby a microcon-50 microconcentrater filter and then subjected to reversetranscription (RT) (20 μl), comprising 2 μl of 10× buffer, 1 μM sensepeptide linker-linked HVk primers of above, dNTPs (1.5 mM each for dATP,dGTP, dCTP and dTTP) and RNase inhibitors (20 U). After M-MuLV reversetranscriptase (40 U) was added, the reaction was incubated at 42° C. for1 hour and shifted to 52° C. for another 15 min. The sense orientationfirst strand cDNAs so obtained were collected by a microcon-50microconcentrater filter. The resulting purified sense orientation firststrand cDNAs were denatured from the antisense RNA transcripts bydenaturation at 94° C. for 3 min and instantly mixed with 1 jiMT7-linked antisense HJk primers mixture as described above. Afterbriefly centrifuging, Taq DNA polymerase (3.5 U) and dNTPs (1.5 mM eachfor dATP, dCTP, dGTP and dTTP) were added and incubated at 52° C. for 3min and then 68° C. for 10 min to form promoter-linked double-strandedcDNAs.

[0200] The resulting ds V_(K) DNA homologs can be furthertranscriptionally amplified by repeating the IVT using T7 RNApolymerase. The resulting ds V_(K) DNA homologs can be the templates forfurther cloning and processing in either an in vivo expression vector orin an in vitro transcription/translation unit system such as thePROfusion or ribosome display method.

[0201] In the case of making an scF_(v) antibody, the V_(K) DNA homologsmade by the steps above can be used as the template for making singlestranded antisense V_(K) sequences by using a single antisense primersequence, such as the antisense Sfi-scFv primer, 5′-GTC CTC GTC GAC TGGAAT TCG GCC CCC GAG GCC AC-3′) (SEQ ID NO:__) (Table 4) in a primerextension reaction. Briefly, the V_(K) DNA homologs (20 ng) made abovewas added to an admixture of 1 μM of the antisense primer above, Taq DNApolymerase (3.5 U) and dNTPs (1.5 mM each for dATP, dCTP, dGTP anddTTP), were combined and the mixture was denatured at 94° C. for 5 min.,then followed by five cycles of 1 min at 94° C., 1 min. at 60° C. and1.5 min at 72° C. The single stranded antisense V_(K) DNA sequences werethen used in an overlapping strand extension reaction with thesingle-stranded sense V_(H) DNA sequences made as described above in areaction as described below.

[0202] The transcriptional amplification of a V_(λ) gene is performedusing the scheme as depicted in FIG. 2. In detail, about 5-10 μg of poly(A)⁺ mRNAs in DEPC-treated water were first hybridized (annealed) with 1μM antisense primer mixture comprising equal amounts of, for example,T7-linked antisense HJ_(λ) primers such as aT7HJ_(λ)-1 (5′-dCCA GTG AATTGT AAT ACG ACT CAC TAT AGG GAA TGG AAT TCG GCC CCC GAG GCC ACC TAG GACGGT GAC CTT GGT CCC-3′) (SEQ ID NO:__); aT7HJ_(λ)-2 (5′-dCCA GTG AAT TGTAAT ACG ACT CAC TAT AGG GAA TGG AAT TCG GCC CCC GAG GCC ACC TAG GAC GGTCAG CTT GGT CCC-3′) (SEQ ID NO:__); and aT7HJ_(λ)-3 (5′-dCCA GTG AAT TGTAAT ACG ACT CAC TAT AGG GAA TGG AAT TCG GCC CCC GAG GCC ACC TAA AAC GGTGAG CTG GGT CCC-3′) (SEQ IN NO:__) at 65° C. for five minutes and cooleddown to room temperature. The reaction was subsequently added to areverse transcription (RT) reaction admixture (20 μl) on ice, comprising2 μl of 10× buffer (400 mM Tris-HCl, pH 8.3, 300 mM KCl, 80 mM MgCl₂, 2M betaine, 100 mM DTT), dNTPs (1.5 mM each for dATP, dGTP, dCTP anddTTP) and RNase inhibitors (20 U). After M-MuLV reverse transcriptase(40 U) was added, the reaction was incubated at 42° C. for 1 hour andshifted to 52° C. for another 15 min. The first strand cDNAs so obtainedwere collected by a microcon-50 microconcentrater filter. The resultingpurified first strand cDNAs were denatured from the mRNAs bydenaturation at 94° C. for 3 min and instantly mixed with 1 μM sense HVλprimers comprising for example, equal amounts of sense peptidelinker-linked HV_(λ) primers, sPLHV_(λ)-1 (5′-TCC TCA GGC GGC GGC GGCTCT GGC GGA GGT GGC AGC GGC GGT GGC GGA TCC CAG TCT GTG TTG ACG CAG CCGCC-3′) (SEQ ID NO:__); sPLHV_(λ)-2 (5′-TCC TCA GGC GGC GGC GGC TCT GGCGGA GGT GGC AGC GGC GGT GGC GGA TCC CAG TCT GCC CTG ACT CAG CCT GC-3′)(SEQ ID NO:__); sPLHV_(λ)-3 (5′-TCC TCA GGC GGC GGC GGC TCT GGC GGA GGTGGC AGC GGC GGT GGC GGA TCC TCC TAT GTG CTG ACT CAG CCA CC-3′) (SEQ IDNO:__); sPLHV_(λ)-4 (5′-TCC TCA GGC GGC GGC GGC TCT GGC GGA GGT GGC AGCGGC GGT GGC GGA TCC TCT TCT GAG CTG ACT CAG GAC CC-3′) (SEQ ID NO:__);sPLHV_(λ)-5 (5′-TCC TCA GGC GGC GGC GGC TCT GGC GGA GGT GGC AGC GGC GGTGGC GGA TCC CAC GTT ATA CTG ACT CAA CCG CC-3′) (SEQ ID NO:__);sPLHV_(λ)-6 (5′-TCC TCA GGC GGC GGC GGC TCT GGC GGA GGT GGC AGC GGC GGTGGC GGA TCC CAG GCT GTG CTC ACT CAG CCG TC-3′) (SEQ ID NO:__); andsPLHV_(λ)-7 (5′-TCC TCA GGC GGC GGC GGC TCT GGC GGA GGT GGC AGC GGC GGTGGC GGA TCC AAT TTT ATG CTG ACT CAG CCC CA-3′) (SEQ ID NO:__). Afterbriefly centrifuging, Taq DNA polymerase (3.5 U) and dNTPs (1.5 mM eachfor dATP, dCTP, dGTP and dTTP) were added to form promoter-linked doublestranded cDNAs at 52° C. for 3 min and then 68° C. for 10 min. An invitro transcription (IVT) reaction (40 μl) was prepared, containing 4 μlof 10× buffer, the above reaction mixture, rNTPs (2 mM each for ATP,GTP, CTP and UTP), and T7 RNA polymerase (160 U). After a 1 hourincubation at 37° C., the antisense amplified V_(λ) RNA transcripts weredirectly used for making the double stranded V_(λ) DNA homologs. Theantisense RNA transcripts were purified and collected by a microcon-50microconcentrater filter and then subjected reverse transcription (RT)by mixing with a reaction admixture (20 μl) comprising 2 μl of 10×buffer, 1 μM sense peptide linker-linked HV_(λ) primers of above, dNTPs(1.5 mM each for dATP, dGTP, dCTP and dTTP) and RNase inhibitors (20 U).After M-MuLV reverse transcriptase (40 U) was added, the reaction wasincubated at 42° C. for 1 hour and shifted to 52° C. for another 15 min.The sense orientation first strand cDNAs so obtained were collected by amicrocon-50 microconcentrater filter. The resulting purified senseorientation first strand cDNAs were denatured from the antisense RNAtranscripts by denaturation at 94° C. for 3 min and then instantly mixedwith 1 μM of the T7-linked antisense HJλ primers mixture as describedabove. After briefly centrifuging, Taq DNA polymerase (3.5 U) and dNTPs(1.5 mM each for dATP, dCTP, dGTP and dTTP) were added and the mixtureincubated at 52° C. for 3 min and then 68° C. for 10 min to formpromoter-linked double stranded cDNAs.

[0203] The resulting ds V_(λ) DNA homologs can be furthertranscriptionally amplified by repeating the IVT using the T7 RNApolymerase. The resulting ds V_(λ) DNA homologs can be the templates forfurther cloning and processing into either an in vivo expression vectoror an in vitro transcription/translation unit mixture such as thePROfusion or ribosome display method.

[0204] In the case of making the scF_(v) antibody, the V_(λ) DNAhomologs made as discussed above can be used as the template for makingsingle stranded antisense V_(λ) sequences by using a single antisenseprimer sequence, such as the antisense Sfi-scFv primer, 5′-GTC CTC GTCGAC TGG AAT TCG GCC CCC GAG GCC AC-3′) (SEQ ID NO:__) (Table 4) in aprimer extension reaction. Briefly, the V_(λ) DNA homologs (20 ng) madeabove were added to an admixture of 1 μM of the above antisense primer,Taq DNA polymerase (3.5 U) and dNTPs (1.5 mM each for dATP, dCTP, dGTPand dTTP), the mixture was denatured at 94° C. for 5 min., then followedby five cycles of 1 min at 94° C., 1 min at 60° C. and 1.5 min at 72° C.The single stranded sense V_(λ) DNA sequences were then used in anoverlapping strand extension reaction with the single stranded senseV_(H) DNA sequences, made above, as follows.

[0205] Linkage of V_(H) and V_(L) into a single chain V_(H)+V_(L)sequence was accomplished using the single stranded sense V_(H) DNAsequences made above and the single stranded sense V_(L) (V_(K) andV_(λ)) made above that were constructed with an overlapping linkersequence that are complementary for hybridization annealing. Theannealed two single stranded sequences can be extended to form dsV_(H)+V_(L) single chain DNA sequences as the scF_(v) DNA homologs (seeFIG. 10) readily for further cloning and expression applications.Briefly, single stranded sense V_(H) DNA sequences (20 ng) made abovewere mixed with 20 ng of single stranded antisense V_(L) (10 ng of V_(K)made above and 10 ng of V_(λ) made above) and added to a solutioncontaining Taq DNA polymerase (3.5 U) and dNTPs (1.5 mM each for dATP,dCTP, dGTP and dTTP), with incubation at 52° C. for 3 min and then 68°C. for 10 min to form ds DNAs. The resulting ds single chainV_(H)-(Gly₄Ser)₃-V_(L) were used in the following cloning steps formaking an scFv antibody phage display library.

[0206] The scF_(v) DNA sequences can be further cloned into anappropriate expression vector or an in vitro transcription/translationreaction mixture. Appropriate restriction site(s) can be added to theflanking sequences for the single chain scF_(v) sequences. In that case,the Sfi cutting site was selected for cloning into a pCGMT9 phage vectoras described by Gao, et al (1999) (Proc. Natl. Acad. Sci. USA 96,6035-6230), see FIG. 10. Briefly, the ds scF_(v) sequences made abovecontaining the Sfi restriction sites at both ends were digested with Sfiand readily ligated with the pCGMT9 vector predigested with Sfi. ThescF_(v) antibody can be expressed and the scF_(v) phage library so madecan be screened for preselected antigen-binding activities according tothe description of Gao et al. (1999) (Proc. Natl. Acad. Sci. USA 96,6035-6230).

Example 11

[0207] Screen of Human Antibody Phage Library for Preselected AntigenBinding Activity

[0208] To identify and to isolate the individual phage clones containingthe human antibody DNA homologs that code for an antigen bindingprotein, the human antibody phage library made as in Examples 6 to 10was titered first and then plated onto agar plates. Replica filter liftswere generated and screened against preselected antigen-binding activityaccording to the manufacturer instruction manual from Stratagene (LaJolla, Calif.).

[0209] The titre of the human antibody expression library preparedaccording to Examples 6 to 10 was determined using methods well known toone skilled in the art and detailed in the instruction manual fromStratagene (La Jolla, Calif.).

[0210] The screening of phage libraries for antigen binding is wellknown in the art. Briefly, the phage plaque nitrocellulose filter liftsof the expressed human antibody in a phage display library were screenedagainst ¹²⁵I-labeled BSA (bovine serum albumin) conjugated with apreselected ligand at a density of approximately 30,000 plaques per 150mm plate. The primary phage plagues identified and isolated weresubjected to a secondary screening.

[0211] Screening employed standard plaque lift methods well known in theart and performed following the instruction manual from Stratagene (LaJolla, Calif.). Typically, the XL1 Blue cells infected with phage wereincubated on 150 mm plates for 4 h at 37° C., protein expression inducedby overlay with nitrocellulose filters soaked in 10 mM isopropylthiogalactoside (IPTG) and the plates incubated at 25° C. for 8 hours.Duplicate filters were obtained during a second incubation employing thesame conditions. Filters were then blocked in a solution of 1% BSA inPBS for 1 hour before incubation with rocking at 25° C. for 1 hour witha solution of ¹²⁵I-labeled BSA conjugated to ligand (2×10⁶ cpm ml⁻¹; BSAconcentration at 0.1 M; approximately 15 ligand molecules per BSAmolecule) in 1% BSA/PBS. Background was reduced by pre-centrifligationof stock radiolabeled BSA solution at 100,000 g for 15 min andpre-incubation of solutions with plaque lifts from plates containingbacteria infected with a phage having no insert. After labeling, filterswere washed repeatedly with PBS/0.05% Tween 20 before development ofautoradiographs overnight.

Example 12

[0212] In Vitro Antibody Expression Libraries Constructed in an In VitroTranscription/Translation Unit

[0213] The V_(H), V_(L) or V_(H)+V_(L) gene repertoire can be expressedin an in vitro transcription and translation system such as thePROfusion system described in the U.S. Pat. No. 6,214,553 to Szostak, etal., Feb. 5, 1999 or in an in vitro transcription and translation systemsuch as the ribosome display system as described in the PCT patentapplication WO 91/05058 by Glenn Kawasaki.

Example 13

[0214] In Vitro Antibody Selection Against Specific Antigen BindingActivity From In Vitro Antibody Expression Libraries.

[0215] The expressed antibody of V_(H), V_(L) or V_(H)+V_(L) generepertoire in an in vitro transcription and translation system such asthe PROfusion system or the ribosome display system can be identifiedand isolated as described in the U.S. Pat. No. 6,214,553 to Szostak, etal. or as described in the PCT patent application WO 91/05058 by GlennKawasaki.

[0216] References

[0217] All references cited herein and herein incorporated by referencein entirety.

[0218] Bird et al.: Science, 242:423-426 (1988).

[0219] Chomczynski et al.: Anal Biochem., 162:156-159 (1987)

[0220] Compton, J.: Nature 350: 91-92 (1991).

[0221] DiLella et al.: Methods In Enzymol., 152:199-212 (1987).

[0222] Eberwine et al.: Proc. Natl. Acad. Sci. USA 89: 3010-3014 (1992).

[0223] Frischauf: Methods In Enzymol., 152:183-190 (1987).

[0224] Frischauf: Methods In Enzymol., 152:190-199 (1987).

[0225] Gao C. S. et al.: Proc. Natl. Acad. Sci. USA 96, 6025-6030(1999).

[0226] Haard H. J. D. et al.: J. Biol. Chem. 274, 18218-18230 (1999).

[0227] Haidaris, C. G. et al.: J. Immunol. Methods 257, 185-202 (2001).

[0228] Herrmann et al.: Methods In Enzymol., 152:180-183, (1987).

[0229] Marks, J. D. et al.: Eur. J. Immunol. 21, 985-991 (1991).

[0230] Marks, J. D. et al.: J. Mol. Biol. 222, 581-597 (1991).

[0231] Methods in Enzymology, Volume 155, pp. 335-350 (1987).

[0232] Murakawa et al.: DNA 7:287-295 (1988).

[0233] Sambrook et.al. “Molecular Cloning, 2nd Edition”,Cold SpringHarbor Laboratory Press, pp8.11-8.19 (1989).

[0234] Lin S.-L. et al.: Nucleic Acid Res. 27: 4585-4589 (1999).

[0235] Welschof, M. et al.: J. Immunol. Methods, 179, 203-214 (1995).

[0236] U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 toMullis et al.

[0237] U.S. Pat. No. 4,704,692 issued to Ladner.

[0238] U.S. Pat. No. 5,130,238 issued to Malek et al.

[0239] U.S. Pat. Nos. 5,409,818; 5,466,586; 5,554,517 and 6,063,603issued to Davey et al.

[0240] U.S. Pat. No. 5,514,545 issued to Eberwine et.al.

[0241] U.S. Pat. No. 5,817,465 issued to Mallet et.al.

[0242] U.S. Pat. No. 5,888,779 issued to Kacian et.al.

[0243] U.S. Pat. No. 5,942,391 issued to Zhang et al.

[0244] U.S. Pat. No. 6,197,554 issued to Lin et.al.

[0245] U.S. Pat. No. 6,214,553 issued to Szostak et al.

[0246] U.S. Pat. No. 6,214,587.

[0247] U.S. Pat. No. 6,287,824 issued to Lizardi.

[0248] U.S. Pat. No. 6,291,158 issued to Winter et al.

[0249] U.S. Pat. No. 6,291,161 issued to Lerner et al.

[0250] EPO Application No. 88113948.9 by Davey & Malek.

[0251] EPO Application No. 89313154 by Kacian & Fultz;

[0252] Europe Patent Publication 320,308.

[0253] PCT patent application WO 91/05058 by Glenn Kawasaki

[0254] WO 88/10315 by Gingeras et al.

[0255] WO 89/1050 by Burg et al.

[0256] WO 91/02818 by Malek et al.

[0257] The ideas, embodiments and examples presented herein provide abetter in vitro RNA transcription-based method and approach toamplification and cloning of the diverse antibody repertoire and theexpression therefrom. The foregoing is intended as illustrative of thepresent invention but not limiting. Numerous variations andmodifications can be effected without departing from the true spirit andscope of the invention.

I claim:
 1. A process for isolating a nucleic acid encoding a proteinthat binds to a target antigen, comprising: (a) exposing a plurality ofnucleic acids encoding a plurality of proteins that bind a plurality ofantigens to at least one primer under conditions suitable to produce aDNA comprising a nucleic acid that functions as an RNA polymerasepromoter; (b) transcribing said DNA of step (a) with a suitable RNApolymerase to produce plural RNAs; (c) optionally, repeating steps (a)and (b) to produce plural copies of said DNA or said RNA; (d) cloningsaid DNA, RNA or functional parts thereof under conditions that enableexpression of said DNA; (e) expressing proteins encoded by said clonedDNA or RNA of step (d); and (f) screening said expressed proteins withsaid target antigen to identify a protein that specifically bindsthereto, thereby identifying a nucleic acid encoding a protein thatbinds to said antigen; wherein when one primer is used, said primercomprises said RNA polymerase promoter, and when two primers are used,either primer comprises said promoter.
 2. The process of claim 1,wherein said nucleic acid is an RNA.
 3. The process of claim 1, whereinsaid protein comprises V_(H), V_(L) or a combination thereof.
 4. Theprocess of claim 3, wherein said protein comprises V_(H) and V_(L). 5.The process of claim 4, wherein said protein further comprises a linkermolecule between said V_(H) and said V_(L).
 6. The process of claim 1wherein said conditions that enable expression comprise a vector in acell, an in vitro transcription/translation reaction mixture or acombination of both.
 7. The process of claim 1, wherein a first primeris used to produce a complement, wherein said complement is treated toyield a 3′ poly C tail and a second primer comprises poly G and saidpromoter.
 8. The process of claim 1 further comprising modifying saidRNA, said DNA or both to yield additional proteins that bind antigen. 9.The process of claim 8, wherein said modifying is by mutagenesis.
 10. Alibrary of proteins, nucleic acids, cells comprising said nucleic acidsor cells expressing said proteins made by the method of claim
 1. 11. Thelibrary of claim 10 comprising at least 10⁴ proteins.
 12. The library ofclaim 11 comprising at least 10⁵ proteins.
 13. The library of claim 12comprising at least 10⁶ proteins.
 14. A process for isolating a nucleicacid encoding a protein that binds to a target antigen comprising: (a)exposing a plurality of nucleic acids encoding different proteins thatbind to different antigens to non-PCR amplification to yield amplifiednucleic acid sequences; (b) cloning said amplified nucleic acidsequences or functional parts thereof in an expression system; (c)expressing proteins encoded by the cloned nucleic acid sequences to forma library of expressed proteins capable of binding different antigens;and (d) identifying a protein that binds to said target antigen, therebyidentifying the nucleic acid encoding said protein that binds to saidtarget antigen.
 15. The process of claim 14, wherein said amplificationcomprises RNA transcription.
 16. The process of claim 14, wherein saidamplification comprises strand displacement.
 17. The process of claim14, wherein said amplification comprises an RNA replicase activity. 18.The process of claim 14, wherein said amplification comprises rollingcircle amplification.
 19. The process of claim 14, wherein the proteinis selected from the group consisting of V_(H), V_(L), and combinationsthereof.
 20. The process of claim 19, wherein the protein comprises aV_(H) and a V_(L) and further comprises a linker bonded between theV_(H) and the V_(L).
 21. The process of claim 14, wherein saidexpression system is selected from the group consisting of an expressionsystem comprising a vector and a host cell, an in vitro transcriptionand translation expression system or a combination thereof; and whereinsaid proteins are expressed using a vector and a host cell, an in vitrotranscription and translation expression system or a combinationthereof.
 22. The process of claim 14 further comprising modifying saidamplified nucleic acid to yield additional proteins that bind antigens.23. The process of claim 22, wherein said modifying is by mutagenesis.24. A library of proteins or nucleic acid sequences encoding saidproteins made by the process of claim
 14. 25. The library of claim 24encoding at least 10⁴ proteins.
 26. The library of claim 25 encoding atleast 10⁵ proteins.
 27. The library of claim 26 encoding at least 10⁶proteins.
 28. A process for making a library of proteins that bind toantigens, comprising: (a) exposing a plurality of nucleic acids encodingdifferent proteins that bind different antigens to at least one primerunder conditions suitable to produce a DNA comprising a nucleic acidthat functions as an RNA polymerase promoter; (b) transcribing the DNAsequence of step (a) with a suitable RNA polymerase to produce pluralRNAs; (c) optionally, repeating steps (a) and (b) to produce pluralcopies of said DNA sequence or said RNA sequence; (d) cloning said DNAsequence or said RNA sequence or functional parts thereof in anexpression system; and (e) expressing proteins encoded by the cloned DNAsequence or RNA sequence or fragment thereof to form a library ofexpressed proteins capable of binding different antigens.
 29. Theprocess of claim 28 further comprising screening the expressed proteinswith an antigen to identify a protein that binds to said antigen. 30.The process of claim 28 wherein said primer comprises said nucleic acidthat functions as an RNA polymerase promoter when one primer is used andwhen two primers are used, either primer comprises the promoter.
 31. Theprocess of claim 28 wherein said nucleic acids are RNA.
 32. The processof claim 28 wherein said proteins are selected from the group consistingof V_(H), V_(L), and combinations thereof.
 33. The process of claim 32wherein said proteins further comprise a linker bonded between the V_(H)and the V_(L).
 34. The process of claim 28 wherein said expressionsystem is selected from a group consisting of an expression systemcomprising a vector and a host cell, an in vitro transcription andtranslation expression system or a combination thereof, and wherein saidproteins are expressed using a vector and a host cell, an in vitrotranscription and translation expression system or a combinationthereof.
 35. The process of claim 28, wherein a first primer is used toproduce a complement, wherein said complement is treated to yield a 3′poly C tail and a second primer comprises poly G and said promotersequence.
 36. The process of claim 28 further comprising modifying saidRNA, said DNA or both to yield additional proteins that bind antigens.37. The process of claim 36, wherein said modifying is by mutagenesis.38. A library of proteins made by the process of claim
 28. 39. Thelibrary of claim 38 encoding at least 10⁴ proteins.
 40. The library ofclaim 39 encoding at least 10⁵ proteins.
 41. The library of claim 40encoding at least 10⁶ proteins.